TWI751110B - Aml antigens and uses thereof - Google Patents

Abstract

The present invention provides novel compounds comprising an antigen of AML cells, and uses thereof.

Description

AML antigens and their uses

The present invention relates to the fields of biology, immunology and medicine.

Acute myeloid leukemia (AML) is a high-risk malignancy with a five-year survival rate of 40% to 50% in patients under the age of 60. For patients older than 65, the results were even worse, with durable remissions in just under 20%. Allogeneic stem cell transplantation is frequently used in the treatment of acute leukemia. It was originally designed to rescue patients from other lethal myeloablative chemotherapy, but was subsequently compromised due to complications associated with an alloreactive immune response (graft versus host disease; GvHD). complication. T-cell deficiency in the graft prior to reinfusion averted GvHD, but T-cell-deficient graft recipients, similar to those of identical twin donors, experienced a higher chance of relapse, making allogeneic It is becoming clear that the success of stem cell transplantation (SCT) depends on the induction of an anti-leukemia immune response (graft versus leukemia (GvL)). This has led to the use of allogeneic stem cell transplantation without myeloablative conditioning (reduced intensity stem cell transplantation (RIST)) strategies to reduce cytotoxicity and make allogeneic SCT available in a larger patient population development, the patient population includes older patients and patients with multiple treatments. The conditioning regimen in RIST is aimed at substantially eliminating the recipient's adaptive immune system to prevent graft rejection without completely removing the recipient's bone marrow, thereby reducing early SCT toxicity. Following transplantation, donor stem cells gradually replace recipient stem cells, and full donor chimerism is typically achieved within three months after SCT. Although allogeneic SCT is effective in the majority of patients and there has been considerable progress in supportive care for SCT recipients, 15-30% of patients still die from transplant-related complications such as GvHD and infectious Complications (due to slow immune recovery after SCT or complications of immunosuppressive therapy for GvHD). Thus, while SCT may be therapeutic when a strong graft-versus-leukemia (GvL) response is elicited, its therapeutic efficacy is limited by the anti-host immune response responsible for GvHD, which results in high morbidity and mortality. Considering the high incidence of GvHD after allogeneic stem cell transplantation, which kills 15-30% of patients, and the fact that a suitable donor is not always available for a given patient, alternative treatment modalities would be helpful of. International patent application PCT/NL2014/050873 provides patient-derived, AML-specific human antibodies that bind to intact AML cells. Importantly, the antibodies were derived from human AML patients who had undergone allogeneic transplantation and had undergone complete remission, showing that the antibodies were effective against AML. The use of antibodies as disclosed in AML therapy as in PCT/NL2014/0508732 is thus preferred.

Instead of passive immunization using antibodies, immunotherapy is also an attractive option. With immunotherapy, a patient with a disease is given a disease-specific antigen, which induces and/or enhances an immune response against the disease in the patient. Prophylactic or semi-prophylactic use, in which an individual is given a disease-specific antigen to elicit an immune response before the disease begins or (further) develops, also attractive. For example, harnessing the immunization of AML-specific target molecules to elicit an immune response against AML would be particularly attractive in patients who have undergone allogeneic hematopoietic stem cell transplantation. The use of this targeted immunization is also attractive for another group of patients with intermediate-high-risk myelodysplastic syndrome (MDS). Such patients are at moderate to high risk of developing AML, so pre-priming an anti-AML immune response would be beneficial. The risk of developing AML in patients with MDS is generally established according to the International Prognosis Scoring System (IPSS; see, for example, Malcovati et al., 2013). Non-limiting examples of patients with intermediate to high risk MDS are MDS-RAEB-1 and MDS-RAEB-2 patients.

Immunotherapies and vaccines specific for AML are currently unavailable due to the lack of suitable AML-specific antigens.

AML-specific antigens are also particularly suitable for determining whether a sample from a patient contains antibodies and/or immune cells that can specifically bind to AML cells. Such information is of considerable value, for example, for the diagnosis of AML or for monitoring AML therapy.

The purpose of this case is to provide novel peptides and compounds comprising antigens of AML cells. Preferably, the provided peptides and compounds are capable of detecting and/or eliciting an immune response, preferably a specific immune response, against myeloproliferative diseases, more preferably against AML.

The present invention provides an isolated, recombinant or purified CD43 peptide having a length of up to 100 amino acid residues, wherein the peptide comprises an amino acid sequence having at least 3 amino acids Residues and lengths and positions of up to 51 amino acid residues are in CD43 amino acid positions as shown in Figure 13 The sequences between 133 and 184 are identical. The peptide preferably comprises an amino acid sequence having at least 3 amino acid residues and at most 51 amino acid residues in length and positioned at the CD43 amine as shown in Figure 13 The sequence between base acid positions 133 and 183 is identical. In some embodiments, the amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues in length residue, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues residue, or at least 25 amino acid residues, or at least 30 amino acid residues, or at least 35 amino acid residues, or at least 40 amino acid residues, or at least 45 amino acid residues residues, or at least 50 amino acid residues, or 51 amino acid residues.

Some embodiments provide an isolated, recombinant or purified CD43 peptide having a length of up to 100 amino acid residues, wherein the peptide includes an amino acid sequence having at least 3 amino acid residues The length of the acid residues and up to 33 amino acid residues is identical to the sequence located between amino acid positions 133 and 165 of CD43 as shown in Figure 13. In some embodiments, the amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues in length residue, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues residues, or at least 25 amino acid residues, or at least 30 amino acid residues, or 33 amino acid residues.

Some embodiments provide an isolated, recombinant or purified CD43 peptide having a length of up to 100 amino acid residues, wherein the peptide comprises a An amino acid sequence having at least 3 amino acid residues and at most 15 amino acid residues in length and located between CD43 amino acid positions 133 and 147 as shown in Figure 13 sequence is the same. In some embodiments, the amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues in length residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or 15 amino acid residues.

Figure 1: Sequence of AT14-013 (2K23-1K13), including variable heavy and light chain sequences and CDR sequences of the antibody.

Figure 2: Binding of AT14-013 to AML cell lines and freshly isolated primary AML blasts from newly diagnosed patients. FAB: French-American-British classification of AML (Bennett et al., 1976).

Figure 3: AT14-013 binding to AML cell lines and primary isolated AML cells. Representative of the binding of AT14-013 derived from patient 101 to AML cell lines Kasumi3, SH-2, Molm13 and THP-1, and to primary leukemia bud cells isolated from newly diagnosed patients (FAB classification M0-M5) Sex example. In-house produced human antibodies specific for influenza virus were used as negative controls (grey filled histogram).

Figure 4: AT14-013 also binds to chronic bone marrow from high-risk myeloid hematopoietic syndromes (MDS/RAEB I and II) and blast crisis Leukemia bud cells from patients with chronic myeloid leukemia (CML). Depicted are representative examples; labeled as patient ID, except for K562, which is a CML cell line. In-house produced human antibodies against influenza virus were used as negative controls (grey filled histogram).

* BL-060: biphenotypic leukemia that responds well to AML therapy.

Figure 5: AT14-013 does not bind to non-myeloid cells. (a) AT14-013 did not bind to healthy PBMCs, T cells (CD3+), B cells (CD19+), non-activated monocytes (CD14+) or primary isolated thymocytes (except for the few present in the fetal thymus). except some bone marrow cells). (b) AT14-013 also did not bind to primary isolated B- or T-ALL cells, lymphoma or multiple myeloma. (c) AT14-013 also did not bind to colon cancer cell lines or primary isolated cells from patients with colon cancer (Colon CA) or healthy colon or ileum.

AT14-013 did bind to granule spheres (a) and to human melanoma cell lines (c). Internally produced human antibodies against influenza virus were used as negative controls (grey filled histogram).

Figure 6: CDC and ADCC. Calcein-labeled THP-1 cells were cultured with AT14-013 and rabbit serum complement. Live cells were identified as calcein+, dapi- cells. With our bead based assay, the amount of dead cells can then be calculated as a measure of complement dependent cell death (CDC). THP1 cells incubated with CD33 did not induce CDC (left). AT14-013 was also able to induce antibody-dependent cell cytotoxicity (ADCC) with the AML cell line SH-2 or freshly isolated leukemia blast cells as target cells in the Jurkat reporter system (right). side).

Figure 7: Target identification of AT14-013: immunoprecipitation (IP). IP of THP1 cell lysate with biotin-labeled (labeled by a sortase) AT14-013 yielded an approximately 140 kDa band on Imperial Coomassie stained colloids. The band was specific because it was not seen in the AT10-002 IP of THP1 cell lysates or in the IP of Jurkat cell lysates. The band was excised from the colloid and the target was identified as CD43 by mass spectrometry analysis.

Figure 8: Target confirmation of AT14-013. THP-1 and Molm13 cell lysates were immunoprecipitated with AT14-013 or the influenza virus specific antibody AT10-002. Western blot analysis with mouse-anti-CD43 (clone Mem59) confirmed CD43 as the binding target of AT14-013.

Figure 9: AT14-013 binds to a unique CD43 epitope. (a) THP-1 cells were stained with commercially available CD43 specific antibodies DF-T1, 84-3C1, L10 and Mem59 and AT14-013. All antibodies bound to the cell membrane of THP-1 cells. (b) AT14-013 has a different binding profile compared to commercially available CD43-specific antibodies. In Kim ea (Kim et al., 2014), the binding of commercially available CD43 antibodies YG5, 2C8, 8E10 and DFT-1 to various cell lines is outlined. We compared the binding of AT14-013 to the same cell lines and found different binding patterns. (c) Competition experiments with AT14-013 and a commercially available CD43-specific antibody were performed as indicated. Briefly, THP-1 cells were incubated with increasing concentrations of the indicated antibodies, after which potentially competing antibodies (referred to as "competing antibodies") were added. Pre-incubation of cells with commercially available CD43 antibody has no effect AT14-013 binds to THP-1 target cells, and these commercially available CD43 antibodies do inhibit the binding of each other to THP-1 cells. The experimental results in which AT14-013 or 84-3C1 are shown as "competing antibodies" are shown. (d) Summary of the competition experiment. AT14-013 did not compete with the commercially available CD43 antibody for binding to THP-1, indicating that AT14-013 binds a different epitope.

Figure 10: Deglycosylation of THP-1 cells to remove sialic acid from the cell membrane with neuraminidase (sialidase). "No" indicates no neuraminidase treatment, "1:20" and "1:200" indicate dilution of neuraminidase. Following neuraminidase treatment of these cells, antibodies AT14-013, Mem 59, DF-T1 and 84-3C1 lost binding to THP-1 cells. Colony L10 did not bind to the sialylated epitope of CD43, and neuraminidase treatment of THP-1 cells did not affect the binding of this antibody to its target cells.

Figure 11: CD43 truncated variants map epitopes to commercially available antibodies DF-T1 and MEM59. a) Immunoblot of HEK293T cells expressing truncated variants of CD43, probed with anti-CD43 against the intracellular C-terminal tail of the protein. b) The same blots were immunostained with CD43-specific antibodies MEM59 (upper) and DF-T1 (lower), revealing that their epitopes were present in region 'C' (amino acids 59-82).

Figure 12: AT14-013 epitope identified from CD43 truncated variants immunoprecipitated from THP1 cells. a) Immunoblotting analysis of input cell lysates of THP1 cells overexpressing sorted CD43 truncated variants, probed with anti-Flag antibodies. PonseauS staining confirmed equal loading of samples. All mutant proteins were expressed. b) Elution immunoprecipitation of THP1 mutant cell lines and anti-Flag immunoblotting analysis of AT14-013 revealed binding to mutants A-F and not to mutants Variants H-J, defining epitopes. c) Immunoblotting analysis with anti-CD43 cytoplasmic tail-binding antibody (Novus) showing endogenous immunoprecipitated CD43 in all samples as well as staining for truncated variants.

Figure 13: Amino acid sequence of CD43 (genbank CCDS10650.1). Signal peptide, AT14-013 epitope, transmembrane domain, and intracellular and extracellular domains are shown.

Figure 14: Binding of AT14-013 to other AML budding cells. Binding of AT14-013 to freshly isolated primary AML bud cells (CD45dim) from newly diagnosed patients. Internally produced human antibodies specific for influenza virus were used as negative controls. For the commercially available mouse anti-CD43 antibody, mouse anti-CMV was used as a control.

WHO: Swerdlow S.H. WHO Classification of Tumors of Hematopoietic and Lymphoid Tissue (2008). CD43+ T cells and tonsil cells were used as additional controls for analysis. AT14-013 was bound to these healthy cells.

(% gated = -; <10%, +; 10~25%, ++; 25~50%, +++; 50~75%, ++++; 75~100%).

Figure 15: ADCC and CDC. a) AT14-013 (open squares) was able to induce antibody dependent cell mediated cytotoxicity (ADCC) in the AML cell line SH-2 and PBMC at an effector-target ratio of 50:1. Live cells were identified as calcein+, dapi- cells. With our bead based assay, the amount of dead cells can then be calculated. SH2 cells incubated with AT10-002 did not induce ADCC (black dots). The calculated EC50 for AT14-013 was 0,16ug/ml. Covered with Calcein-labeled SH-2 cells (b) with AT14-013 (open squares) or AT10-002 (black dots) were incubated with rabbit serum complement. Live cells were identified as calcein+, dapi- cells. SH2 cells or AML budding cells incubated with AT10-002 did not induce CDC (black dots). The calculated EC50 for AT14-013 was 1,86ug/ml.

Figure 16: a) Elution immunoprecipitation of THP1 variant cell lines and anti-Flag immunoblot analysis of AT14-013 revealed binding to mutants A-F2, less binding to G and no binding to mutants H-J. b) Immunoblotting analysis with anti-CD43 cytoplasmic tail-binding antibody (Novus) showing endogenous immunoprecipitated CD43 in all samples as well as staining for truncated variants. This control group was determined to be successful in immunoprecipitation in all samples shown.

Figure 17: a) Treatment of mice engrafted with SH-2 AML cells resulted in 90.3% inhibition of tumor growth as measured by whole body measurements at sacrifice (p<0.001, repeated one-way analysis of variance (ANOVA)) . b) The number of AML cells, measured by photons per minute (cpm), showed a substantial reduction in all measured organs (p=0.0011, 2way ANOVA). c) The number of tumor cells was assessed by FACS in bone marrow and liver (p=0.0017, two-way analysis of variance).

Figure 18: Representative example of AT14-013 binding to fetal CD34+ hematopoietic stem cells (HSCs) but not to fetal CD34+CD38+ precursor cells or fetal CD34-CD38- mature cells. Grey filled histogram: control antibody against influenza virus AT10-002, as described in WO 2013/081463).

Figure 19: AT14-013 reacts with autologous leukemia stem cells. Donor #101's AML budding cells (same donor from which AT14-013-producing B cells were obtained) were specific for AT14-013 and for CD34 and CD38 , and with an antibody against CD45 (BD, cat 348815) were stained to identify the overall blast cell population (CD45 dim) and healthy cells in the bone marrow and analyzed by flow cytometry.

The inventors of the present invention have surprisingly found that a specific immune response against AML can be detected and/or elicited using the CD43 peptide of the present invention. This finding is surprising since CD43 is present on the surface of most types of (non-malignant) white blood cells, but also on many non-hematopoietic tumor cells, such as human colon cancer cells, human cervical cancer cells, human lung cancer cells, and human breast cancer cells, etc. In view of such abundant presence of CD43, prior to the present invention, CD43 was not considered a suitable compound to provide specificity for AML. However, as shown in the Examples, the AML-specific antibody AT14-013 binds the CD43 peptide of the present invention. In addition, antibody AT14-013 bound to a variety of different CD43+ AML cells (panels 2 and 3), but not CD43+ PBMC, activated and non-activated T cells, B cells, non-activated monocytes, thymocytes, ALL cells, colon cancer cells, non-malignant colon cells, Jurkat cells, Ramos cells or normal bone marrow cells (as shown in Figure 5). Thus, the antibody AT14-013, which is specific for the CD43 peptide as defined in the scope of the claims, binds to CD43+ AML cells, but not to various other types of CD43+ cells. Interestingly, antibody AT14-013 did not bind to other species of non-AML, CD43+ hematopoietic stem cells or more mature cells of the lymphoid lineage. The examples also show that antibody AT14-013 can bind to the oncofetal epitope of CD43, antibody AT14-013 can bind autologous leukemia stem cells, and antibody AT14-013 can combat AML growth in vivo. The present invention thus provides the CD43 antigen of AML cells.

CD43, also known as leukosalin, sialophorin, galactoglycoprotein, leukosialoglycoprotein, or gp115, is a glycosylated mucin-like protein ) type I transmembrane protein, which is present on the surface of most hematopoietic cells, with the exception of red blood cells. CD43, encoded by an exon, plays a role in cell-cell interactions. It has a highly glycosylated extracellular region of 235 amino acids. Two CD43 glycoforms have been described, one glycoform containing mainly four-carbon sugars, and the other glycoform having mainly branched six-carbon sugars. Both glycoforms can be expressed on the surface of a cell. CD43 is described, for example, in Shelley et al. (1989) and Schmid et al. (1992). The sequence of human CD43, as shown in Figure 13, exists in the Genbank CCDS database under accession number CCDS10650.1.

As used herein, the term "CD43 peptide of the present invention" refers to an amino acid chain having a length of up to 100 amino acid residues, wherein the amino acid chain includes a sequence of at least 3 The amino acid residues and up to 51 amino acid residues in length are identical to the sequence between amino acid positions 133 and 184 of the human CD43 protein as shown in Figure 13, or wherein said amino acid The chain includes a sequence having a length of at least 3 amino acid residues and at most 51 amino acid residues and a sequence located between amino acid positions 133 and 183 of the human CD43 protein as shown in Figure 13 the same, or wherein the amino acid chain comprises a sequence of at least 3 amino acid residues and at most 33 amino acid residues in length and positioned at the amino groups of the human CD43 protein as shown in Figure 13 The sequence between acid positions 133 and 165 is identical, or wherein the amino acid chain comprises a sequence of at least 3 amino acid residues and at most 15 amino acid residues in length and position as shown in Figure 13 The sequence shown is identical between amino acid positions 133 and 147 of the human CD43 protein.

As explained in detail in the Examples, the present invention provides insight that the amino acid sequence between positions 133 and 184 of the human CD43 protein includes an AML epitope that is specifically bound by the antibody AT14-013. The AML epitope includes one or more amino acid residues present between positions 133 and 165 of the amino acid sequence shown in FIG. 13 . The AML epitopes, which are present on different AML cell lines and AML budding cells and which are absent or exposed on many other CD43+ cells, are therefore particularly suitable for use in eliciting or detecting AML-specific immune responses. In some embodiments, the CD43 peptide of the invention comprises the amino acid sequence GTITTNSPETSSRTS. In some embodiments, the inventive CD43 peptide comprises the amino acid sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTS.

In some embodiments, the inventive CD43 peptide comprises the amino acid sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSLETSKGTSG.

In some embodiments, the CD43 peptides of the invention are up to 90 amino acid residues in length. In some embodiments, the CD43 peptides of the invention have a length of up to 85 amino acid residues, or up to 75 amino acid residues, or up to 70 amino acid residues. In some embodiments, the CD43 peptide of the invention has at most 65 amino acid residues, or at most 60 amino acid residues, or at most 55 amino acid residues, or at most 50 amino acid residues groups, or up to 45 amino acid residues, or up to 40 amino acid residues, or up to 35 amino acid residues in length. In some embodiments, the CD43 peptide of the present invention has at most 52 amino acid residues, or at most 51 amino acid residues, or at most 33 amino acid residues amino acid residues, or up to 30 amino acid residues, or up to 25 amino acid residues, or up to 20 amino acid residues, or up to 15 amino acid residues in length. In some embodiments, the CD43 peptide of the present invention has at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues group, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues in length.

In some embodiments, the CD43 peptide of the present invention has a length of at least 52 amino acid residues or at least 51 amino acid residues, wherein the peptide comprises an amino acid sequence that is linked to the position of The sequence between amino acid positions 133 and 184 of the human CD43 protein as shown in Figure 13 is identical. In some embodiments, the CD43 peptide of the present invention has a length of at least 51 amino acid residues, wherein the amino acid residues are the same as the amino acid positions of the human CD43 protein as shown in FIG. 13 . The amino acids between 133 and 183 are the same.

In some embodiments, the inventive CD43 peptide has a length of at least 33 amino acid residues and includes an amino acid sequence that is linked to amino acid position 133 of the human CD43 protein as shown in Figure 13 and the sequence between 165 is the same.

In some embodiments, the inventive CD43 peptide has a length of at least 15 amino acid residues and includes an amino acid sequence that is linked to amino acid position 133 of the human CD43 protein as shown in Figure 13 and the sequence between 147 is the same.

In some embodiments, the inventive CD43 peptide is represented by the sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTSGPPLTMATVSL Composed of ETSK GTSG.

In some embodiments, the inventive CD43 peptide consists of the sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTS.

In some embodiments, the inventive CD43 peptide consists of the sequence GTITTNSPETSSRTS.

As used herein, the words "sequence located between amino acid positions X and Y as shown in Figure 13", "positioned at amino acid position X of the human CD43 protein as shown in Figure 13 and Sequence between Y", "wherein said amino acid residue is identical to the amino acid between amino acid positions X and Y of the human CD43 protein as shown in Figure 13", and "monoamino acid sequence" , which is identical to the sequence located between amino acid positions X and Y of the human CD43 protein as shown in Figure 13 "encompassing amino acids located between the recited positions and including positions X and/or Y sequence. Furthermore, the term includes sequences of amino acids between the recited positions and excluding positions X and/or Y. In other words, in some embodiments, the recited amino acids at positions X and/or Y are present in the CD43 peptides of the invention, while in other embodiments, the recited amino acids at positions X and/or Y are present in the CD43 peptides of the invention. does not exist.

Except for sequences between amino acid positions 133 and 184 of the human CD43 protein as shown in Figure 13, or between amino acid positions 133 and 183, or between amino acid positions 133 and 165 The CD43 peptides of the present invention may further comprise other amino acid residues in addition to the recited amino acid sequences in which the sequences between amino acid positions 133 and 147 are identical. In some embodiments, the other amino acid residues are not derived from the CD43 sequence. Such other amino acid residues, also referred to herein as "non-CD43 amino acid residues", may function, for example, to enhance stability, and/or enhance immunogenicity, and/or couple the CD43 peptide to another moiety, such as a molecular scaffold or carrier. Non-limiting examples of such scaffolds or carriers are keyhole limpet hemocyanin and CLIPS scaffolds (eg, bis(bromomethyl)benzene, tris(bromomethyl)benzene, and tetra(bromomethyl) benzene, as described in WO 2004/077062). Some embodiments thus provide an isolated, recombinant or purified peptide having a length of up to 100 amino acid residues, wherein the peptide comprises an amino acid sequence having at least 3 amino acid residues the length of amino acid residues and up to 52 amino acid residues or up to 51 amino acid residues is identical to the sequence located between amino acid positions 133 and 184 of CD43 as shown in Figure 13, and wherein the peptide also includes at least 1, or at least 2, or at least 3, or at least 4, or at least 5, or at least 10, or at least 20, or at least 30, or at least 40 , or at least 50, or at least 60, or at least 70, or at least 80 non-CD43 amino acid residues, wherein the full-length sequence of said non-CD43 amino acid residues is not present as shown in Figure 13 in human CD43.

Some embodiments provide an isolated, recombinant or purified peptide having a length of up to 100 amino acid residues, wherein the peptide comprises an amino acid sequence having at least 3 amines The length of amino acid residues and up to 51 amino acid residues is identical to the sequence between amino acid positions 133 and 183 of CD43 as shown in Figure 13, and wherein the peptide also includes at least one , or at least 2, or at least 3, or at least 4, or at least 5, or at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60 , or at least 70, or at least 80 non-CD43 amino acid residues, wherein the full-length sequence of said non-CD43 amino acid residues is not present in human CD43 as shown in FIG. 13 . In some embodiments, the amine The length of the amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues, or at least 12 amino acid residues amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues, or at least 25 amines amino acid residues, or at least 30 amino acid residues, or at least 35 amino acid residues, or at least 40 amino acid residues, or at least 45 amino acid residues, or at least 50 amines amino acid residues, or at least 51 amino acid residues.

Some embodiments provide an isolated, recombinant or purified peptide having a length of up to 100 amino acid residues, wherein the peptide comprises an amino acid sequence having at least 3 amines The length of amino acid residues and up to 33 amino acid residues is identical to the sequence between amino acid positions 133 and 165 of CD43 as shown in Figure 13, and wherein the peptide also includes at least one , or at least 2, or at least 3, or at least 4, or at least 5, or at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60 , or at least 70, or at least 80 non-CD43 amino acid residues, wherein the full-length sequence of said non-CD43 amino acid residues is not present in human CD43 as shown in FIG. 13 . In some embodiments, the amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues in length acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues acid residues, or at least 25 amino acid residues, or at least 30 amino acid residues, or at least 33 amino acid residues.

Some embodiments provide an isolated, recombinant or purified peptide having a length of up to 100 amino acid residues, wherein the peptide comprises an amino acid sequence having at least 3 amines The length of amino acid residues and up to 15 amino acid residues is identical to the sequence between amino acid positions 133 and 147 of CD43 as shown in Figure 13, and wherein the peptide also includes at least one , or at least 2, or at least 3, or at least 4, or at least 5, or at least 10, or at least 20, or at least 30, or at least 40, or at least 50, or at least 60 , or at least 70, or at least 80 non-CD43 amino acid residues, wherein the full-length sequence of said non-CD43 amino acid residues is not present in human CD43 as shown in FIG. 13 . In some embodiments, the amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues in length acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues.

The above-mentioned peptides are also included in the term "CD43 peptide of the present invention".

Some embodiments provide a compound comprising a CD43 peptide of the present invention. Some embodiments provide an immunizing compound comprising a CD43 peptide of the present invention. In some embodiments, the CD43 peptide is linked to a pharmaceutically acceptable carrier or scaffold.

In some embodiments, the inventive CD43 peptides are truncated CD43 molecules having a length of up to 100 amino acid residues. Preferably, the truncated CD43 molecule lacks the intracellular region of wild type human CD43. In preferred embodiments, the truncated CD43 molecule lacks both the intracellular and transmembrane regions of wild-type human CD43. in more In a preferred embodiment, the CD43 peptide of the present invention is a truncated CD43 extracellular region, which has at most 90 amino acid residues, or at most 80 amino acid residues, or at most 70 amino acid residues. acid residues, or up to 60 amino acid residues, or up to 52 amino acid residues, or up to 51 amino acid residues, or up to 50 amino acid residues, or up to 45 amino acid residues acid residues, or up to 40 amino acid residues, or up to 35 amino acid residues, or up to 33 amino acid residues, or up to 30 amino acid residues, or up to 25 amino acid residues acid residues, or up to 20 amino acid residues in length, the truncated CD43 extracellular region comprising an amino acid sequence having at least 3 amino acid residues and at most 52 amino acid residues amino acid residues or up to 51 amino acid residues in length identical to the sequence located between amino acid positions 133 and 184 of CD43 as shown in Figure 13. In some embodiments, the amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues in length acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues acid residues, or at least 25 amino acid residues, or at least 30 amino acid residues, or at least 35 amino acid residues, or at least 40 amino acid residues, or at least 45 amino acid residues acid residues, or at least 50 amino acid residues, or at least 51 amino acid residues.

In some embodiments, the CD43 peptide of the invention is a truncated CD43 extracellular region having at most 90 amino acid residues, or at most 80 amino acid residues, or at most 70 amino acid residues residues, or up to 60 amino acid residues, or up to 51 amino acid residues, or up to 50 amino acid residues, or up to 45 amino acid residues, or up to 40 amino acid residues residues, or up to 35 amino acid residues, or up to 33 amino acid residues, or up to 30 amino acid residues base, or up to 25 amino acid residues, or up to 20 amino acid residues in length, the truncated CD43 extracellular region includes a monoamino acid sequence having at least 3 amino acid residues and up to 51 amino acid residues in length identical to the sequence located between amino acid positions 133 and 183 of CD43 as shown in Figure 13. In some embodiments, the amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues in length acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues acid residues, or at least 25 amino acid residues, or at least 30 amino acid residues, or at least 35 amino acid residues, or at least 40 amino acid residues, or at least 45 amino acid residues acid residues, or at least 50 amino acid residues, or at least 51 amino acid residues.

In some embodiments, the CD43 peptide of the invention is a truncated CD43 extracellular region having at most 90 amino acid residues, or at most 80 amino acid residues, or at most 70 amino acid residues residues, or up to 60 amino acid residues, or up to 51 amino acid residues, or up to 50 amino acid residues, or up to 45 amino acid residues, or up to 40 amino acid residues residue, or up to 35 amino acid residues, or up to 33 amino acid residues, or up to 30 amino acid residues, or up to 25 amino acid residues, or up to 20 amino acid residues The length of the residues, the truncated CD43 extracellular region includes an amino acid sequence having at least 3 amino acid residues and at most 33 amino acid residues in length and position as shown in Figure 13 The sequence shown is identical between amino acid positions 133 and 165 of CD43. In some embodiments, the amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues in length amino acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues, or at least 20 amino acid residues amino acid residues, or at least 25 amino acid residues, or at least 30 amino acid residues, or at least 33 amino acid residues.

In some embodiments, the CD43 peptide of the invention is a truncated CD43 extracellular region having at most 90 amino acid residues, or at most 80 amino acid residues, or at most 70 amino acid residues residues, or up to 60 amino acid residues, or up to 51 amino acid residues, or up to 50 amino acid residues, or up to 45 amino acid residues, or up to 40 amino acid residues residue, or up to 35 amino acid residues, or up to 33 amino acid residues, or up to 30 amino acid residues, or up to 25 amino acid residues, or up to 20 amino acid residues The length of the residues, the truncated CD43 extracellular region includes an amino acid sequence of at least 3 amino acid residues and at most 15 amino acid residues in length and position as shown in Figure 13 The sequence shown is identical between amino acid positions 133 and 147 of CD43. In some embodiments, the amino acid sequence is at least 5 amino acid residues, or at least 8 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues in length acid residues, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues.

Some embodiments provide a CD43 peptide according to the invention, which is a truncated CD43 extracellular region having up to 90 amino acid residues, or up to 80 amino acid residues, or up to 70 amino acid residues amino acid residues, or up to 60 amino acid residues, or up to 52 amino acid residues, or up to 51 amino acid residues, or up to 50 amino acid residues, or up to 45 amino acid residues group, or up to 40 amino acid residues, or up to 35 amino acid residues, or up to 33 amino acid residues residues, or up to 30 amino acid residues, or up to 25 amino acid residues, or up to 20 amino acid residues, or up to 15 amino acid residues in length.

As is known to the skilled person, once the immunogenic sequence has been provided, it becomes possible to alter the sequence to some extent, and thus preferably optimize the immunogenicity and/or stability of the resulting immunogen. This can be done, for example, by a mutagenesis step in which the stability and/or immunogenicity of the resulting compound is preferably tested, and an improved AML-specific antigenic compound is selected. The skilled artisan is well able to generate antigenic variants starting from specific amino acid sequences. For example, conservative amino acid substitutions are used. Examples of conserved amino acids include substitution of a hydrophobic residue, such as isoleucine, valine, leucine, or methionine, for another hydrophobic residue, and substitution of one polar residue for another Polar residues such as arginine to lysine, glutamic acid to aspartic acid, or glutamic acid to aspartic acid. In some embodiments, a replacement net analysis is performed, which involves the replacement of one or more amino acid residues with any other amino acid residue, and the resulting compounds are tested.

Some embodiments thus provide an isolated, recombinant or purified CD43 peptide having a length of up to 100 amino acid residues comprising or consisting essentially of an amino acid sequence having at least 3 amino acid residues and up to 52 amino acid residues or 51 amino acid residues in length and the sequence between amino acid positions 133 and 184 of the human CD43 protein as shown in Figure 13 have at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity . Some embodiments provide an isolated, recombinant or purified CD43 peptide comprising an amino acid sequence having 51 amino acid residues The length of the amino acid sequence is at least 80%, preferably at least 85%, preferably at least 90% of the sequence between amino acid positions 133 and 183 of the human CD43 protein as shown in Figure 13 , preferably at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity.

Some embodiments provide an isolated, recombinant or purified CD43 peptide having a length of up to 100 amino acid residues comprising or consisting essentially of an amino acid sequence having 33 amines The length of the amino acid residues is at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 80%, with the sequence between amino acid positions 133 and 165 of the human CD43 protein as shown in Figure 13 at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity.

Some embodiments provide an isolated, recombinant or purified CD43 peptide having a length of up to 100 amino acid residues comprising or consisting essentially of an amino acid sequence having 15 amines The length of amino acid residues is at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 80%, with the sequence between amino acid positions 133 and 147 of the human CD43 protein as shown in Figure 13 at least 95%, preferably at least 96%, preferably at least 97%, preferably at least 98%, preferably at least 99% sequence identity.

The term "% sequence identity" is defined herein as the candidate amino acid sequence after aligning the two sequences, the candidate amino acid sequence and the reference sequence, and introducing gaps, if necessary, to achieve maximum percent identity. Percentage of residues in the acid sequence that are identical to residues in the reference sequence. Methods and computer programs for calibration are well known in the art. One available or suitable computer program for determining whether a candidate sequence falls within this definition is "Align 2", developed by Genentech, Prepared by Inc., which was filed with user documentation on December 10, 1991, U.S. Copyright Office, Washington D.C., 20559.

An isolated, recombinant or purified CD43 peptide as defined herein is also referred to as "inventive CD43 peptide" or "inventive CD43 antigen". In some embodiments, the amino acid residues of the inventive CD43 peptides are selected from 20 amino acid residues that occur naturally in eukaryotic cells, also referred to as "standard" or "canonical" amino acid residues acid. Alternatively, inventive CD43 peptides comprise unnatural amino acid residues, such as D-amino acids (ie, D-stereoisomers of amino acids) or N-methyl amino acids.

The CD43 peptides of the invention are preferably glycosylated. Such peptides more accurately reflect native AML antigens in vivo, since native CD43 protein is highly glycosylated on the cell surface. In some preferred embodiments, the CD43 peptides described herein include sialic acid residues (also known as α-N-acetylneuraminic acid). In vivo, human CD43 is highly sialylated. Treatment with neuraminidase cleaves sialic acid residues from the CD43 glycoprotein. In some embodiments, therefore, the inventive CD43 peptides are neuraminidase-sensitive. Some embodiments provide CD43 peptides of the invention that are onco-sialylated, representing that the CD43 peptides have a tumor-specific sialylated form. As used herein, an inventive CD43 peptide having a "tumor-specific sialylated form" encompasses an inventive CD43 peptide having a sialylated form produced by tumor cells, or having the same sialylated form as produced by tumor cells, or At least 90%, or at least 95%, or at least 97% similar sialylated forms of the inventive CD43 peptides.

Some embodiments provide CD43 peptides of the invention, which are produced by AML cells. Some embodiments provide CD43 peptides of the invention, which are derived from Produced from cells of a cell line of AML cells. Such CD43 peptides will have a glycosylated form that is very similar to that of AML cells in AML patients. In some embodiments, the CD43 peptide of the invention is produced by THP-1 cells. In some embodiments, the CD43 peptide of the invention is produced by Kasumi 3 cells, or by HL60 cells, or by KG1a cells, or by SH2 cells, or by MonoMac6 cells, or by Molm13 cells, or by CML K562 cells .

As used herein, a CD43 peptide of the invention has a glycosylation form that is similar or identical to that produced by the expression of the CD43 peptide in AML cells (eg, in AML bud cells or AML cell lines) The CD43 peptide of the invention is said to have an AML-specific glycosylation form. Some embodiments thus provide CD43 peptides of the invention having an AML-specific glycosylation form.

Some embodiments provide CD43 peptides of the invention, which are produced by MDS cells. Some embodiments provide CD43 peptides of the invention produced by cells of a cell line derived from MDS cells. Such CD43 peptides will have a glycosylated form that is very similar to the glycosylated form of MDS cells in MDS patients.

Some embodiments provide inventive CD43 peptides having MDS-specific glycosylation forms. These peptides have a glycosylated form that is similar or identical to the glycosylated form produced by expressing the inventive CD43 peptides in MDS cells (eg, in MDS bud cells or MDS cell lines).

In other embodiments, CD43 peptides of the invention are provided which are produced by host cells using in vivo glycoengineering (eg according to Roche Diagnostics GmbH). According to these embodiments, a host is provided The cells have the enzymes α-2,6-sialyltransferase and/or α-2,3-sialyltransferase, so that after the inventive CD43 peptide is produced, the peptide will be sialylated. Therefore, the present invention further provides a CD43 peptide, which is produced by cells comprising α-2,6-sialyltransferase and/or α-2,3-sialyltransferase. In some embodiments, the α-2,6-sialyltransferase and/or α-2,3-sialyltransferase comprises exogenous α-2,6-sialyltransferase and/or Alpha-2,3-sialyltransferase, indicating that the enzyme has been recombinantly introduced into the host cell (or into the parent host cell from which the current host cell originated). Some embodiments provide CD43 peptides of the invention produced by host cells comprising exogenous nucleic acid sequences encoding alpha-2,6-sialyltransferase and/or alpha-2,3-sialyltransferase.

Anti-CD43 antibodies are known in the art. However, it is clear that these antibodies recognize different epitopes. For example, as shown in Table 1 of Kim et al. (2014), the anti-CD43 monoclonal antibodies (mAbs) YG5, 2C8, 8E10 and DFT-1 do bind AML cells, but many other non-AML cells, including CEM7, Jurkat, IM9 , Ramos, Raji, Daudi, Reh, normal bone marrow and PBL cells were also bound by some or all of these known antibodies (Kim et al., 2014). Therefore, the antibodies YG5, 2C8, 8E10 and DFT-1 were all specific for AML degeneration. In contrast, antibody AT14-013, which specifically binds CD43 of the present invention, did not bind Jurkat cells, Ramos cells, normal bone marrow cells or PBL cells/PBMCs (Figures 5 and 9b). Antibodies YG5, 2C8, 8E10 and DFT-1 thus bind another CD43 epitope.

Antibody UN1 (Tuccillo et al., 2014a and Tuccillo et al., 2014b) recognizes a CD43 epitope comprising a GalNac-O-linked monosaccharide, corresponding to the Tn antigen of the O polysaccharide. The conclusion reached is that this antigenic decision The core protein of the base contains CD43 amino acids 64 to 83. This antigen is expressed by human thymocytes, by the leukemia cell lines HPB-ALL, H9 and Molt-4, and in a subpopulation of peripheral blood CD4+ T lymphocytes. Antibody AT14-013, however, did not bind human thymocytes, indicating that the present invention provides a different AML antigen.

International patent application WO 2007/146172 describes antibodies 5F1, 51-41 and 138-10 which recognize CD43 present on the surface of the human colorectal adenocarcinoma cell line Colo205 and the human gastric cancer cell line NCI-N87. AML is not mentioned in WO 2007/146172. Antibody AT14-013, which specifically binds the AML-specific CD43 peptide of the present invention, does not bind Colo205, as shown in the Examples. Antibodies 5F1, 51-41 and 138-10 thus bind different CD43 epitopes.

International patent application WO 2006/121240 describes antibodies EB-1, EB-2 and EB-3 capable of recognizing the unglycosylated region of CD43, which consists of CD43 amino acids 73-81. This antigen is present on thymocytes, on some hematopoietic precursors in the bone marrow, on AML cells, on acute lymphogenous leukemia (ALL) cells, and on chronic myelogenous leukemia (CML) on cells. EB-1 recognizes its antigen in both sialidase-treated and non-sialidase-treated CD43 molecules. On the contrary, after treatment with sialidase (neuraminidase), the glycosylated CD43 of the present invention is no longer recognized by the antibody AT14-013, which means that the present invention provides an AML antigen that is sensitive to neuraminidase. , in contrast to the antigens of EB-1, EB-2 and EB-3. Furthermore, antibody AT14-013 did not bind to ALL cells or thymocytes, in contrast to antibodies EB-1, EB-2 and EB-3. Furthermore, the CD43 peptide of the present invention includes an amino acid sequence having at least 3 amino acid residues, at least 3 amino acid residues and at most 51 amino acid residues in length and an amino group at CD43. The sequences within acid residues 133 and 184 are identical, and their domains differ from amino acid residue positions 73 to 81 of CD43 recognized by antibodies EB-1, EB-2 and EB-3. Antibodies EB-1, EB-2 and EB-3 thus also bind another CD43 epitope.

In summary, the present invention provides a novel AML-specific antigen.

The CD43 peptide of the invention preferably has at most 100 amino acid residues and at least 3 amino acid residues, preferably at least 5 amino acid residues, or at least 6 amino acid residues, or At least 7 amino acid residues, or at least 8 amino acid residues, or at least 9 amino acid residues, or at least 10 amino acid residues, or at least 11 amino acid residues in length group, or at least 12 amino acid residues, or at least 13 amino acid residues, or at least 14 amino acid residues, or at least 15 amino acid residues in length. In some embodiments, the length is at least 20 amino acid residues. In some embodiments, the length is at least 25 amino acid residues. In some embodiments, the length is at least 30 amino acid residues. In some embodiments, the length is at least 33 amino acid residues. In some embodiments, the length is at least 35 amino acid residues. In some embodiments, the length is at least 40 amino acid residues. In some embodiments, the length is at least 45 amino acid residues. In some embodiments, the length is at least 50 amino acid residues. In some embodiments, the length is at least 51 amino acid residues. In some embodiments, the length is at least 52 amino acid residues.

In some embodiments, the length is up to 90 amino acid residues group, or up to 85 amino acid residues, or up to 75 amino acid residues, or up to 70 amino acid residues, or up to 65 amino acid residues, or up to 60 amino acid residues group, or up to 55 amino acid residues, or up to 52 amino acid residues, or up to 51 amino acid residues, or up to 50 amino acid residues, or up to 45 amino acid residues group, or up to 40 amino acid residues, or up to 35 amino acid residues, or up to 30 amino acid residues, or up to 25 amino acid residues, or up to 20 amino acid residues base. In some embodiments, the peptide has at most 52 amino acid residues and at least 3 amino acid residues, preferably at least 5 amino acid residues, or at least 6 amino acid residues, Or at least 7 amino acid residues, or at least 8 amino acid residues, or at least 9 amino acid residues, or at least 10 amino acid residues in length. In some embodiments, the peptide has at most 51 amino acid residues and at least 3 amino acid residues, preferably at least 5 amino acid residues, or at least 6 amino acid residues, Or at least 7 amino acid residues, or at least 8 amino acid residues, or at least 9 amino acid residues, or at least 10 amino acid residues in length. In some embodiments, the peptide has at most 33 amino acid residues and at least 3 amino acid residues, preferably at least 5 amino acid residues, or at least 6 amino acid residues, Or at least 7 amino acid residues, or at least 8 amino acid residues, or at least 9 amino acid residues, or at least 10 amino acid residues in length. In some embodiments, the peptide has at most 15 amino acid residues and at least 3 amino acid residues, preferably at least 5 amino acid residues, or at least 6 amino acid residues, Or at least 7 amino acid residues, or at least 8 amino acid residues, or at least 9 amino acid residues, or at least 10 amino acid residues in length.

In some embodiments, the inventive CD43 peptides consist of a single amino acid sequence having at least 3 amino acid residues and at most 51 amino acid residues Or up to 51 amino acid residues in length identical to the sequence between CD43 amino acid positions 133 and 183 as shown in Figure 13. In some embodiments, the inventive CD43 peptide consists of an amino acid sequence having a length of at least 5 amino acid residues and at most 40 amino acid residues and positions as shown in Figure 13 The sequence between CD43 amino acid positions 133 and 183 is identical. In some embodiments, the inventive CD43 peptide consists of an amino acid sequence having a length of at least 5 amino acid residues and at most 33 amino acid residues and the positions shown in FIG. 13 The sequence between CD43 amino acid positions 133 and 183 is identical. In some embodiments, the inventive CD43 peptide consists of an amino acid sequence having a length of at least 5 amino acid residues and at most 20 amino acid residues and the positions shown in FIG. 13 The sequence between CD43 amino acid positions 133 and 183 is identical.

In some embodiments, the inventive CD43 peptide consists of an amino acid sequence having a length of at least 5 amino acid residues and at most 33 amino acid residues and the positions shown in FIG. 13 The sequence between CD43 amino acid positions 133 and 165 is identical. In some embodiments, the inventive CD43 peptide consists of an amino acid sequence having a length of at least 5 amino acid residues and at most 30 amino acid residues and positions as shown in Figure 13 The sequence between CD43 amino acid positions 133 and 165 is identical. In some embodiments, the inventive CD43 peptide consists of an amino acid sequence having a length of at least 5 amino acid residues and at most 20 amino acid residues and the positions shown in FIG. 13 The sequence between CD43 amino acid positions 133 and 165 is identical.

In some embodiments, the inventive CD43 peptide consists of an amino acid sequence having a length of at least 5 amino acid residues and at most 15 amino acid residues and the positions shown in FIG. 13 At amino acid positions 133 and 147 of CD43 The sequence between is the same. In some embodiments, the inventive CD43 peptide consists of an amino acid sequence having a length of at least 8 amino acid residues and at most 15 amino acid residues and the positions shown in FIG. 13 The sequence between CD43 amino acid positions 133 and 147 is identical. In some embodiments, the inventive CD43 peptide consists of an amino acid sequence having a length of at least 10 amino acid residues and at most 15 amino acid residues in the positions shown in FIG. 13 The sequence between CD43 amino acid positions 133 and 147 is identical.

In some embodiments, the above-mentioned peptides are glycosylated, preferably including sialic acid residues, in order to better mimic natural AML-specific antigens. In some embodiments, the aforementioned peptides are onco-sialylated. In some embodiments, the above-mentioned peptides are produced by AML cells or AML cell lines, preferably THP-1 cells. In some embodiments, the above-described peptides are produced by MDS cells or MDS cell lines.

The present invention also encompasses nucleic acid molecules encoding the inventive CD43 peptides or functional equivalents thereof. There is therefore further provided an isolated, synthetic or recombinant nucleic acid molecule or functional equivalent thereof encoding the CD43 peptide of the invention. As used herein, a nucleic acid molecule or nucleic acid sequence of the invention preferably comprises a chain of nucleotides, more preferably DNA, cDNA or RNA. In other embodiments, inventive nucleic acid molecules or nucleic acid sequences include other types of nucleic acid structures, such as DNA/RNA helices, peptide nucleic acids (PNA), locked nucleic acids (LNA), and/or ribozymes. Such other nucleic acid structures are referred to as functional equivalents of nucleic acid sequences. The term "functional equivalents of nucleic acid molecules" thus encompasses chains comprising non-natural nucleotides, modified nucleotides and/or non-nucleotide building blocks that exhibit the same function as natural nucleotides.

Some embodiments provide inventive nucleic acid molecules, or functional equivalents thereof, wherein the human nucleic acid sequences have been codon-optimized for non-human cells, e.g., non-human producer cells such as E. coli , Chinese Hamster Ovary (CHO) cells , NSO cells (which are mouse myeloma) or 293(T) cells. This represents that one or more codons from the human nucleic acid sequence have been replaced with one or more codons preferred by the non-human cell.

As used herein, an isolated, synthesized or recombinant nucleic acid molecule or functional equivalent thereof encoding an inventive CD43 peptide is also referred to as an "inventive nucleic acid molecule or functional equivalent".

Since the invention has provided CD43 peptides comprising novel AML-specific antigens, many applications have become possible. For example, in some embodiments, the CD43 peptides of the invention are used to induce, isolate and/or obtain immune cells and/or antibodies, or functional parts or functional derivatives thereof, which can specifically bind to lymphocyte proliferation and / or myeloproliferative cells. Immune cells and/or antibodies induced, isolated and/or obtained with the inventive CD43 peptides, or functional parts or functional derivatives thereof, are particularly suitable for the treatment or prevention of myeloproliferative or lymphoproliferative diseases. Furthermore, in view of the fact that the antibody AT14-013, which is an antibody specific for the CD43 peptide of the present invention, also targets leukemia stem cells, which are known to be more resistant to therapy and are often diseased after therapy cause of recurrence. In some embodiments, the inventive CD43 peptides are used to induce and/or obtain AML-specific immune cells and/or AML-specific antibodies. For example, with one or more CD43 peptides of the invention, or with immunizing compounds comprising the CD43 peptides of the invention, or with nucleic acid molecules encoding the CD43 peptides of the invention or functional equivalents thereof, or with the invention A nucleic acid molecule or a functionally equivalent vector to immunize non-human animals, and more One or more booster administrations are preferably followed. Next, immune cells and/or antibodies specific for lymphoproliferative and/or myeloproliferative cells, preferably specific for AML, are collected from the non-human animal. In some embodiments, the immune cells comprise T cells, eg, natural killer cells (NK cells) or T helper cells.

In some embodiments, the immune cells collected from the immunized non-human animal comprise B cells, eg, AML-specific B cells are particularly suitable for the production of AML-specific antibodies. AML-specific B cells collected from the immunized non-human animal, for example, can be used for the generation of hybridomas from which AML-specific antibodies are obtained. In other embodiments, B cells collected from the immunized animal are transduced with Bcl-6 and Bcl-xL nucleic acids, and cultured in B cell cultures long-term ex vivo, e.g. , as described in European Patent No. 1974017 and US Patent No. 9,127,251. Here, a culture of long-term replicating B cells is made in which the B cells replicate and produce antibodies. In some embodiments, the AML-specific antibodies produced by the fusionoma or such B cell cultures are collected and, for example, used in anti-AML therapy, preferably after humanization of the antibody ( after humanization). In some embodiments, antibodies and/or B cells obtained from the non-human animal are tested for competition with antibody AT14-013 for CD43 binding. This is done, for example, by culturing AML cells with the antibody or B cells obtained from the non-human animal, and then adding the antibody AT14-013. As a control, AML cells are preferably cultured with the antibody AT14-013 in the absence of any other antibodies or B cells. If preincubation of AML cells with antibodies or B cells derived from the non-human animal appeared to affect AT14-013 binding to the AML cells, then it was concluded that the antibodies derived from the non-human animal Or B cells compete with the antibody AT14-013 for binding to CD43.

In some embodiments, the variable domains of nucleic acid sequences encoding B cells from the non-human animal are sequenced to obtain nucleic acid sequences of AML-specific variable domains comprising one or more of these sequences The nucleic acid sequence of AML is used to generate AML-specific antibodies after introduction into producer cells, such as E. coli , Chinese Hamster Ovary (CHO) cells, NSO cells or 293(T) cells. The one or more nucleic acid sequences are preferably codon-optimized for the producer cell. As used herein, the term "codon" refers to a triplet of nucleotides (or functional equivalents thereof) that encode a particular amino acid residue. The term "codon optimized" means that one or more codons from the original, animal nucleic acid sequence are preferred by one or more codons from a cell from another species (eg, a particular producer cell) replaced. These substituted codons preferably encode the same amino acid residues as the original animal codons being substituted.

In some embodiments, the CD43-specific antibody obtained from the non-human animal or from immune cells of the non-human animal is humanized and represents at least a portion of an animal's amino acid sequence, preferably At least a part or all of the framework sequence is replaced by a human sequence to reduce adverse side effects in humans.

Animal immunization protocols with suitable administration procedures and adjuvants, procedures for obtaining and purifying antibodies and/or immune cells from such immunized animals, competition experiments and humanization procedures for non-human antibodies are well known in the art . For example, Hanly et al. (1995) may be referred to.

In some embodiments, the CD43 peptides of the present invention or compounds comprising the CD43 peptides of the present invention are used to screen phage display libraries to identify and/or isolate AML-specific immunoglobulins (usually Fabs). fragment). In some embodiments, naïve phage display libraries are used. In a preferred embodiment, a phage display library derived from one or more AML patients is used such that the display library will be predisposed to AML. In some embodiments, AML-specific immunoglobulins obtained from the phage display library are tested for competition with antibody AT14-013 for CD43 binding. This is done, for example, using the competition test described herein.

Therefore, there is further provided the use of the inventive CD43 peptide, or the use of the inventive immunizing compound, or the use of the CD43 peptide encoding the inventive CD43 peptide, or the use of a vector comprising the inventive nucleic acid molecule or functional equivalent for induction, isolation and /or the use of obtaining immune cells or antibodies, or functional parts or functional derivatives thereof such as Fab fragments. The immune cells, or antibodies, or functional parts or functional equivalents thereof are preferably capable of specifically binding to lymphoproliferative cells and/or myeloproliferative cells. Preferably, the myeloproliferative cells are AML cells, MDS cells and/or CML cells, most preferably AML cells. In some embodiments, an inventive CD43 peptide, or an inventive immunological compound, or an inventive nucleic acid molecule or functional equivalent, or a vector comprising an inventive nucleic acid molecule or functional equivalent is used to induce and/or obtain a Antibodies that induce antibody-dependent cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). A non-limiting example of such an antibody is AT14-013, as shown in the Examples. The fact that the inventive CD43 peptide or the inventive immunizing compound can induce and/or acquire antibodies with ADCC and/or CDC inducing activity means that the antibodies that can be induced or acquired are effective in vivo .

Also provided herein is the use of inventive CD43 peptides or compounds as immunogens, and the use of inventive nucleic acid molecules or functional equivalents, or vectors comprising inventive nucleic acid molecules or functional equivalents, as immunogens.

Some embodiments provide a method for generating immune cells and/or antibodies capable of specifically binding to lymphoproliferative cells and/or myeloproliferative cells, such as AML-specific immune cells or AML-specific Antibodies, the method comprising making a non-human with the CD43 peptide of the invention, or with the compound of the invention, or with the nucleic acid molecule or functional equivalent of the invention, or with a vector comprising the nucleic acid molecule or functional equivalent of the invention Animal immunity. The method preferably further comprises collecting from the non-human animal immune cells and/or antibodies capable of specifically binding to lymphoproliferative cells and/or myeloproliferative cells. In some embodiments, AML-specific immune cells and/or AML-specific antibodies are collected from the non-human animal. In some embodiments, B cells and/or antibodies obtained from a non-human animal are tested for competition with antibody AT14-013 for CD43 binding. Also provided herein are immune cells and/or antibodies that can specifically bind to lymphoproliferative cells and/or myeloproliferative cells, obtainable by the methods of the present invention. Some embodiments provide AML-specific antibodies or AML-specific immune cells obtainable by the methods of the invention for generating AML-specific immune cells or AML-specific antibodies. Such AML-specific antibodies preferably compete with antibody AT14-013 for binding to CD43.

The non-human animals preferably include mammals, such as rodents or cattle. In some embodiments, the non-human animals include mice, rats, rabbits, llamas, camels, pigs, poultry, cattle, goats, horses, apes, and / or gorilla.

Given the fact that antibody AT14-013 specifically binds the CD43 peptide of the invention, other antibodies obtained, produced or selected with the CD43 peptide of the invention will generally compete with antibody AT14-013 for binding to CD43. In contrast, the existing CD43 antibodies known in the art did not compete with the antibody AT14-013, and these known antibodies did compete with each other, as shown in FIG. 9 . Thus these other antibodies known in the art do indeed bind an epitope different from that of AT14-013. Accordingly, provided further are isolated, recombinant or purified antibodies, or functional portions or functional equivalents thereof, that compete with antibody AT14-013 for binding to CD43. Said isolated, recombinant or purified antibody, or functional part or functional equivalent thereof, preferably competes with antibody AT14-013 for binding to amino acid 133 at the CD43 sequence as shown in Figure 13 and At least a portion of the epitope between 184. The isolated, recombinant or purified antibody, or functional portion or functional equivalent thereof, typically competes with antibody AT14-013 for binding to the CD43 peptide of the invention.

The term "antibody" as used herein refers to an immunoglobulin comprising two heavy chains bound to each other, wherein each heavy chain is also paired with a light chain.

A "functional portion of an antibody" is defined herein as a portion that shares at least one characteristic with the antibody, qualitatively, but not necessarily quantitatively. The functional moiety is capable of binding the same antigen as the antibody, although not necessarily to the same extent. The functional portion of the antibody preferably includes at least one heavy chain variable domain (VH) and one light chain variable domain (VL). In some embodiments, the functional portion of the antibody includes at least one heavy chain variable domain (VH). Non-limiting examples of functional parts of antibodies are single domain antibodies, single chain antibodies, nanobodies, unibodies, single chain variable fragments (scFv), bispecific T cell adapters (bi- specific T-cell engager, BiTE), Fab fragments and F(ab') ₂ fragments.

A "functional equivalent of an antibody" is defined herein as an artificial binding compound comprising at least one CDR sequence of an antibody, preferably a heavy chain CDR3 sequence. The functional equivalents preferably include the heavy chain CDR3 sequences of the antibody and the light chain CDR3 sequences of the antibody. More preferably, the functional equivalents include the heavy chain CDR1, CDR2 and CDR3 sequences of the antibody and the light chain CDR1, CDR2 and CDR3 sequences of the antibody. Functional equivalents of antibodies are generated, for example, by altering the antibodies such that at least one antigen-binding characteristic of the resulting compound is substantially identical in nature, but not necessarily in quantity. This can be done in a number of ways, such as by conservative amino acid substitutions, in which one amino acid residue is replaced by another residue with substantially similar characteristics (size, hydrophobicity, etc.), such that the overall effect of the antibody is substantially is not affected. A non-limiting example of a functional equivalent of an antibody is an antibody with a modified Fc tail, wherein the Fc tail is modified, eg, by amino acid substitutions and/or changes in glycosylation.

As is well known to the skilled artisan, the heavy chain of an antibody is the larger of the two types of chains that make up an immunoglobulin molecule. Heavy chains include constant domains and variable domains, where the variable domains are involved in antigen binding. The light chain of an antibody is the smaller of the two types of chains that make up an immunoglobulin molecule. Light chains include constant and variable domains. The variable domain is usually, but not always, involved in antigen binding together with the variable domain of the heavy chain.

Complementarity determining regions (CDRs) are hypervariable regions present in the heavy and light chain variable domains. In the case of whole antibodies, CDRs 1-3 of the heavy chain and CDRs 1-3 of the linked light chain together form the antigen binding site.

As used herein, an immune cell, antibody, or functional part thereof A compound or functional equivalent is "specific" for AML if it can bind with a higher binding affinity to said AML cells than an irrelevant control antibody or control immune cell (wherein the control antibody or control immune cell Not specific for said AML cells) binds AML cells with at least two-fold binding affinity. For example, AML binding of AML-specific antibodies, B cells, or T cells is typically modulated by antibodies, B cell receptors (BCRs), or T cell receptors (TCRs), respectively. The specific three-dimensional structure of the variable domains of both the AML antigen and antibody or BCR or TCR allows these two structures to bind together (similar to a lock and key interaction) that interacts with any, non-specific, antibody or BCR or TCR Sexual adhesion is the opposite. However, some reactivity to other types of cells is included in the term "AML-specific". Since antibodies or BCRs or TCRs generally recognize epitopes of antigens, and since such epitopes may also be present on other cells, AML-specific antibodies, B cells or T cells may then recognize other cells. Non-limiting examples of such other cells are MDS and CML cells. Melanoma cells and melanocytes also appear to be bound by the AML-specific antibody AT14-013, albeit to a lesser extent. Thus, the term "AML-specific" does not exclude binding of an antibody or B cell or T cell to another cell comprising at least a portion of the same epitope. However, it is shown in the examples that many CD43+ cells, including CD43+ hematopoietic cells such as PBMCs, (pioneer) T cells and B cells, do not contain the AML antigen provided by the present invention.

The CD43 peptides of the present invention are particularly suitable for detecting the presence of AML-specific binding compounds, such as AML-specific antibodies or AML-specific immune cells (eg, B cells or T cells), in biological samples. For example, a sample from an individual, or a fraction of such a sample comprising antibodies, B cells and/or T cells, is combined with CD43 of the invention, or a compound comprising CD43 of the invention Incubate together to check for the presence of AML-specific antibodies and/or AML-specific immune cells. If such antibodies or immune cells are shown to be present in the sample or the sample fraction and bind to the CD43 peptide of the present invention, the sample is classified as specifically binding to a compound for AML (ie, an antibody and/or immune cells) were positive. Such samples include, for example, blood samples, or bone marrow samples, or biological specimens such as myeloid sarcoma (also known as chloroma).

AML-specific antibodies or AML-specific immune cells are, for example, detected and/or quantified using immunoassays, such as Western blot analysis, (capture) ELISA or RIA. These assays are well known in the art. The labeled CD43 peptide of the invention is incubated, for example, with a blood sample or a bone marrow sample, or with a biological specimen such as myeloid sarcoma, or with a fraction of such a sample comprising antibodies, B cells and/or T cells, Unbound bound compound is then washed away. Next, it was determined whether the inventive CD43 peptide was bound by AML-specific antibodies or immune cells. In some embodiments, an unlabeled inventive CD43 peptide, or an unlabeled compound comprising an inventive CD43 peptide, is contacted with a sample comprising antibodies and/or immune cells, such as a blood sample, Or a bone marrow sample, or a biological specimen such as myeloid sarcoma, or a fraction of such a sample that includes antibodies, B cells and/or T cells. After incubation, one or more washing steps are preferably performed to remove unbound antibodies and unbound immune cells. Next, it is tested whether the antibody or immune cell has bound to the CD43 peptide of the invention, for example, an antibody specific for human antibodies or human immune cells and linked to a label, such as a fluorescent compound or horseradish catalase, can be used (horseradish peroxidase) or alkaline phosphatase. After a further washing step, it is determined whether the secondary antibody has been bound, for example by measuring the emitted light or adding Mt. A substrate for catalase or alkaline phosphatase. These detection techniques are well known in the art.

In some embodiments, an inventive CD43 peptide, or a compound comprising an inventive CD43 peptide, is contacted with a fraction of a sample already enriched for antibodies and/or immune cells. In some embodiments, the fraction is an in vitro culture of B cells or an in vitro culture of T cells. In some embodiments, an inventive CD43 peptide or a compound comprising an inventive CD43 peptide is contacted with antibodies and/or immune cells that have been substantially purified from a biological sample, such as purified B cells obtained by selecting CD19 positive cells Fractions and/or antibody/B cell fractions purified using anti-Ig antibody or protein A or G purification methods. Protein A or G purification methods are well known in the art, and method schemes and reagents are commercially available. As used herein, the term "immune cells that have been substantially purified from a sample" means that at least 80%, preferably at least 85%, more preferably at least 90% or at least 95% of the cells of the produced fraction are composed of immune cells . The term "antibody that has been substantially purified from a sample" means that at least 80%, preferably at least 85%, more preferably at least 90% or at least 95% of the mass of the produced fraction consists of antibody.

Thus further provided is the use of the inventive CD43 peptide, or a compound comprising the inventive CD43 peptide, for binding and/or detecting immune cells, and/or antibodies, or functional parts or functional equivalents thereof. The immune cells, and/or antibodies, or functional parts or functional equivalents thereof are preferably capable of specifically binding to lymphoproliferative cells and/or myeloproliferative cells. Preferably, the lymphocyte proliferative cells are AML cells, or MDS cells, or CML cells, preferably AML cells. Also provided herein are inventive CD43 peptides, or compounds comprising inventive CD43 peptides, as detection moieties for AML-specific binding compounds use of the AML-specific binding compounds such as antibodies and/or immune cells, and to provide a method for determining whether a sample includes AML-specific antibodies and/or AML-specific immune cells, the method comprising adding the inventive CD43 to The peptide, or a compound comprising the CD43 peptide of the invention, is incubated with the sample, or a fraction of the sample comprising antibodies and/or immune cells, and then it is determined whether the CD43 peptide of the invention is affected by AML-specific antibodies and/or AML-specific immune cells, or whether the compound comprising the CD43 peptide of the invention is bound by AML-specific antibodies and/or immune cells. If such binding is detected, it is concluded that the sample includes AML-specific antibodies and/or AML-specific immune cells.

Also provided is a method for determining whether a sample includes AML-specific antibodies and/or AML-specific immune cells, the method comprising combining an inventive CD43 peptide, or a compound comprising an inventive CD43 peptide, with substantially Purified antibodies and/or immune cells are incubated together, and then it is determined whether the CD43 peptide of the invention is bound by the AML-specific antibody and/or AML-specific immune cells, or the compound comprising the CD43 peptide of the invention Whether bound by AML-specific antibodies and/or immune cells.

In some embodiments, the results of a detection test as described above are used to determine whether an individual has AML. If a sample from an individual is shown to contain AML-specific immune cells and/or AML-specific antibodies, it is concluded that the individual is an AML patient. Accordingly, the use of the inventive CD43 peptides as diagnostic reagents and the use of compounds comprising the inventive CD43 peptides as diagnostic reagents are also provided herein. Further provided are the use of the inventive CD43 peptide for diagnosing AML, and the compound comprising the inventive CD43 peptide for diagnosing AML the use of. Further provided is a diagnostic kit comprising: - the CD43 peptide of the invention, or a compound comprising the CD43 peptide of the invention, and - a tool for detecting antibodies that bind the CD43 peptide or immune cells that bind the CD43 peptide.

Such tools encompass, for example, labeled antibodies that are specific for human antibodies or human immune cells. In some embodiments, the labeled antibody is conjugated to horseradish catalase or alkaline phosphatase.

Some embodiments provide a diagnostic kit comprising: - an inventive CD43 peptide, or a compound comprising an inventive CD43 peptide, and - a tool for detecting antibodies or immune cells.

Such tools encompass, for example, labeled antibodies that are specific for human antibodies or human immune cells. In some embodiments, the labeled antibody is conjugated to horseradish catalase or alkaline phosphatase.

Some embodiments provide a method for classifying a sample comprising an antibody or a sample comprising an immune cell, the method comprising adding an inventive CD43 peptide (optionally in the context of an MHC complex to detect T cells), or Contacting a compound comprising the CD43 peptide of the invention with the antibody and/or immune cells of the sample, and determining whether the CD43 peptide of the invention, or the compound of the invention is affected by the antibody and/or immune cell of the sample At least one of the cells binds. If the CD43 peptide or the compound of the invention is bound by antibodies and/or immune cells of the sample, the sample is classified as comprising CD43-specific antibodies and/or immune cells.

Some embodiments provide a method for determining whether an individual has myeloproliferative lymphocyte proliferative disease, preferably AML, the method comprising adding the inventive CD43 peptide (optionally in the context of MHC complexes to detect T cells), or comprising the inventive CD43 peptide The compound is contacted with antibodies and/or immune cells of the individual, and it is determined whether the CD43 peptide of the invention, or the compound of the invention, is bound by at least one of the antibodies and/or immune cells of the individual. If the CD43 peptide or the compound described in the invention is bound by the antibody and/or immune cells of the individual, it is concluded that the individual has a lymphoproliferative or myeloproliferative disease, such as AML. In some embodiments, the CD43 peptide of the invention, or the compound comprising the CD43 peptide of the invention, is contacted with a sample comprising antibodies and/or immune cells of the individual, such as a blood sample , or a bone marrow sample, or a biological specimen such as myeloid sarcoma. In other embodiments, the CD43 peptide or compound of the invention is contacted with a fraction of a sample from the individual, wherein the fraction includes immune cells and/or antibodies. In some embodiments, the CD43 peptide or compound of the invention is contacted with antibodies and/or immune cells that have been substantially purified from the sample, eg, a purified B cell fraction obtained by selecting CD19 positive cells and/or Or antibody/B cell fraction purified by anti-Ig antibody or protein A or G purification method.

In some embodiments, the results of the inventive detection tests are used to determine whether an individual exhibits a detectable immune response against a myeloproliferative or lymphoproliferative disease (eg, AML). For example, this is preferably used to determine whether a patient with a myeloproliferative disorder who has been treated with a drug has a GvL response, such as an AML patient who has been treated for AML, such as an AML patient who has undergone immunotherapy such as stem cell transplantation or donation AML patients with lymphocyte infusion. To date, no patients have been tested for the presence of a strong GvL response in treated patients. break tool. This type of diagnostic tool is highly needed because: 1) it will allow early identification of allogeneic SCT recipients at high risk of relapse, at a point in time before relapse, and thus allow for early intervention, such as immunosuppressive therapy; Reduced or donor-lymphocyte infusion; 2) it will allow such donor lymphocyte infusions to be titrated until anti-leukemia antibodies do appear; and 3) when the presence of a strong GvL response can be verified, it will be administered often subject to many Hope for allogeneic SCT recipients suffering from SCT-related complications. Patients today have to wait and see if relapse occurs, and there is no way to predict disease relapse. Therefore, the availability of tests to determine whether a patient has an anti-AML immune response will greatly improve the clinical care of SCT patients, affecting prognosis and quality of life.

Some embodiments thus provide a method for determining whether an individual exhibits an immune response against a myeloproliferative or lymphoproliferative disease, preferably AML, the method comprising adding an inventive CD43 peptide, optionally to an MHC complex. contacting the antibody and/or immune cells of the individual under an environment, or the compound comprising the CD43 peptide of the invention, and measuring the CD43 peptide of the invention, or the compound comprising the CD43 peptide of the invention Is bound by at least one of the antibody and/or immune cells of the individual. If the CD43 peptide of the invention or the compound is shown to be bound, it means that the individual exhibits an immune response against the myeloproliferative or lymphoproliferative disease (preferably AML). In some embodiments, it is determined whether the individual's antibody or B cell competes with antibody AT14-013 for binding to CD43. Competing antibodies will be particularly effective against myeloproliferative or lymphoproliferative diseases, preferably AML.

In some embodiments, an antibody that competes with antibody AT14-013 for binding to CD43, or a functional portion or functional equivalent thereof, is used to detect in a sample myeloproliferative cells. Thus further provided is the use of an isolated, recombinant or purified antibody, or a functional part or functional equivalent thereof, that competes with the antibody AT14-013 for binding to CD43 for determining whether a sample comprises myeloproliferative cells, preferably myeloproliferative cells For AML or MDS or CML cells. Also provided herein are isolated, recombinant or purified antibodies, or functional portions or functional equivalents thereof, that compete with the antibody AT14-013 for binding to CD43, for use in lymphoproliferative or myeloproliferative diseases (preferably AML or MDS) or CML), and the use of an isolated, recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with the antibody AT14-013 for binding to CD43, for the diagnosis of AML. An isolated, recombinant or purified antibody, or functional portion or functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43 is also provided for use in the preparation of lymphoproliferative or myeloproliferative cells (preferably AML or MDS or CML cells) the use of diagnostic reagents. Some embodiments provide a diagnostic kit comprising an isolated, recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43, and a diagnostic kit for detecting antibody-cell complexes. tool. Such tools include, for example, another antibody against AML cells, such as AT14-013. In some embodiments, the means comprise a labeled antibody directed against another cell surface component of the bone marrow cells. In some embodiments, the means comprise a labeled antibody against the antibody or functional portion or functional equivalent that competes with antibody AT14-013.

Also provided is the use of an isolated, recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with the antibody AT14-013 for binding to CD43 as a diagnostic reagent. Some embodiments provide methods for determining whether myeloproliferative cells are present in a sample, comprising: - Allow the sample to compete with the antibody AT14-013 for binding to CD43 contacting the isolated, recombinant or purified antibody, or a functional part or functional equivalent thereof, and - allowing said antibody or functional part or functional equivalent to bind myeloproliferative cells, if present, and - assaying myeloproliferative cells Binding to the antibody or functional moiety or functional equivalent determines the presence or absence of myeloproliferative cells in the sample. In some embodiments, the myeloproliferative cells are AML or MDS or CML cells.

Further provided is the use of antibody AT14-013, or a functional portion or functional equivalent thereof, for determining whether a sample comprises AML or MDS or CML cells. Also provided herein are the use of antibody AT14-013, or a functional part or functional equivalent thereof, in the diagnosis of AML or MDS or CML, and the use of antibody AT14-013, or a functional part or functional equivalent thereof in the diagnosis of AML or use of MDS or CML. Also provided is the use of antibody AT14-013, or a functional portion or functional equivalent thereof, for the manufacture of a diagnostic reagent for AML or MDS or CML.

Other interesting applications of the novel CD43 peptides and nucleic acid molecules of the invention or functional equivalents encoded thereby are prophylactic or semi-prophylactic applications and immunotherapy. As used herein, semi-prophylactic use means that an individual already has a disease, but further progression of the disease is at least temporarily delayed or prevented. For example, the CD43 peptides of the invention, or nucleic acid molecules or functional equivalents encoded thereby, or vectors comprising the nucleic acid molecules or functional equivalents of the invention can be semi-prophylactically administered to patients with moderate to high Individuals at risk for myelodysplastic syndrome (MDS). As mentioned earlier, such patients are at moderate to high risk of developing AML and thus pre-elicit an anti-AML immune response Should it be beneficial to the patient, the inventive CD43 peptide or compound or nucleic acid molecule or functional equivalent or vector may be utilized prior to progression of MDS to AML. Another example of semi-prophylactic use of the CD43 peptides of the invention, or compounds or nucleic acid molecules or functional equivalents or vectors of the invention, is in AML, MDS or CML patients undergoing allogeneic hematopoietic stem cell transplantation (HSCT). the use of. The goal of such allogeneic HSCT is to elicit an allogeneic graft-versus-leukemia/MDS response, but there is currently no way to confirm the development of such a response. The present invention provides CD43 peptides of the invention, or nucleic acid molecules or functional equivalents encoded thereby, or vectors comprising the nucleic acid molecules or functional equivalents of the invention, or compounds comprising the CD43 peptides of the invention, as inducers for expression Use of prophylactic or semi-prophylactic agents for the alloreactive immune response of CD43 malignant cells (graft versus tumor response, eg, against MDS, AML or CML). Another example of prophylactic or semi-prophylactic use of the CD43 peptides of the invention, or compounds or nucleic acid molecules or functional equivalents or vectors of the invention, is its use in CML patients. Currently, CML is well controlled in many patients using tyrosine kinase inhibitors such as Imatinib. However, patients may develop resistance to one or more tyrosine kinase inhibitors. In addition, the use of tyrosine kinase inhibitors is sometimes associated with adverse side effects such as edema, skin rash, fatigue, nausea, and myelosuppression. Tyrosine kinase inhibitors are also expensive. The present invention provides CD43 peptides of the invention, or nucleic acid molecules or functional equivalents encoded thereby, or vectors comprising the nucleic acid molecules or functional equivalents of the invention, or compounds comprising the CD43 peptides of the invention, as prophylactic or Use of a semi-prophylactic agent to delay or prevent the progression of CML to AML. In some embodiments, the CD43 peptide or compound or nucleic acid molecule or functional equivalent or carrier of the invention is used instead of tyrosine kinase Enzyme inhibitors, for example, to reduce side effects and/or cost. In other embodiments, the CD43 peptides or compounds or nucleic acid molecules or functional equivalents or vectors of the invention are used together with one or more tyrosine kinase inhibitors. Accordingly, also provided herein are inventive CD43 peptides, or compounds comprising inventive CD43 peptides, or nucleic acid molecules encoding inventive CD43 peptides or functional equivalents thereof, or comprising inventive nucleic acid molecules or functional equivalents The carrier of the drug, the use as a prophylactic or semi-prophylactic agent. Also provided are inventive CD43 peptides, or compounds comprising inventive CD43 peptides, or nucleic acid molecules or functional equivalents encoding the inventive CD43 peptides, or vectors comprising inventive nucleic acid molecules or functional equivalents, using Use in the preparation of prophylactic or semi-prophylactic agents against AML, eg, in AML patients who have undergone allogeneic HSCT. In some embodiments, the semi-prophylactic agent is for MDS or CML patients, as explained above. The prophylactic or semi-prophylactic agent preferably includes a vaccine. As used herein, the term "prophylactic agent" also includes semi-prophylactic agents.

In some embodiments, the inventive CD43 peptide or compound or nucleic acid molecule or functional equivalent or vector is used to treat myeloproliferative or lymphoproliferative diseases, preferably AML. As used herein, "treating" includes alleviating at least one symptom and/or delaying or even halting the progression of the disease, at least temporarily. In a preferred embodiment, the CD43 peptide of the invention, optionally in the context of an MHC complex, or a nucleic acid molecule or functional equivalent encoded thereby, or a nucleic acid molecule or functional equivalent comprising the invention. The carrier, or compound comprising the CD43 peptide of the invention, is administered to an AML patient to boost his/her immune system, resulting in an enhanced immune response. In some embodiments, naïve T cells or B cells from AML patients are cultured ex vivo and cultured with inventive CD43 peptides or compounds, optionally complexed in MHC in the case of T cell cultures AML-specific T cells or B cells can be obtained in the context of an organism and then administered to a patient, optionally after expansion in vitro. In some embodiments, it is determined whether AML-specific B cells from the AML patient compete with antibody AT14-013 for binding to CD43, as competing B cells will be particularly effective against AML, especially considering that AT14-013 also targets leukemia The fact that stem cells are known to be more resistant to treatment and is often responsible for disease recurrence after treatment.

In some embodiments, adoptive cell therapy is used. In some embodiments, T cells from AML patients are tested for binding or activation using an inventive CD43 peptide in the context of an MHC complex, or using a compound comprising an inventive CD43 peptide in the context of an MHC complex , and T cells recognizing the CD43 peptide are preferably expanded ex vivo and then administered to a patient, which will result in an anti-AML T cell response.

In some embodiments, cell infusion therapy of donor lymphocytes is used. Donor T cells are isolated from AML patients who have undergone allogeneic HSCT, or from HSCT donors, preferably using the CD43 peptide of the invention in the context of MHC complexes, or using the invention in the context of MHC complexes Compounds of the CD43 peptide are tested for binding or activation, and donor T cells recognizing the CD43 peptide are preferably expanded ex vivo and then administered to the patient, which will result in anti-AML allogeneic T cells reaction.

In some embodiments, T cells are modified to provide their AML-specific binding moiety. The T cells are preferably derived from AML patients or MDS patients or CML patients or HSCT donors. In some embodiments, chimeric antigen receptor (CAR) T cells are generated. These T cells with modified T cell receptors, which have been provided with the binding specificity of the person of interest, are preferably derived from antibodies. Generally, CAR T cells are generated by fusing a single-chain variable domain (scFv) derived from a monoclonal antibody to the CD3-ζ (zeta) transmembrane domain, so that zeta signaling will be elicited upon target recognition by the scFv .

According to some embodiments, inventive CD43 peptides, or nucleic acid molecules or functional equivalents encoded thereby, or vectors comprising inventive nucleic acid molecules or functional equivalents, or compounds comprising inventive CD43 peptides are used to CD43-specific B cells and/or antibodies are produced and/or isolated, which in turn are used in the production of modified T cells. For example, the CD43 peptide or compound or nucleic acid molecule or functional equivalent or vector is used to elicit, detect and/or isolate AML-specific antibodies or AML-specific B cells. Next, in some embodiments, the heavy and/or light chain variable domains of the antibody or B cell are provided to T cells, thereby producing modified T cells specific for AML. In some embodiments, these modified T cells are then administered to an AML patient, which will result in an AML-specific T cell response. In some embodiments, the modified T cells are CAR T cells. In some embodiments, the AML-specific antibody or AML-specific B cell is tested for competition with antibody AT14-013 for CD43 binding after providing the antibody or B cell's heavy and/or light chain variable domains at before T cells. Such competing antibodies are preferably selected for the production of modified T cells specific for AML.

Therefore, the CD43 peptide of the invention is further provided, optionally in the context of MHC complexes, or a compound comprising the CD43 peptide of the invention, or a nucleic acid molecule encoding the CD43 peptide of the invention or a functional equivalent thereof , or a carrier comprising an inventive nucleic acid molecule or functional equivalent for use as a drug. Inventive CD43 peptides are also provided, optionally in the context of the MHC complex For the production of AML Use of specific T cells. Some embodiments provide a method for generating modified T cells, the method comprising contacting an antibody comprising a sample from an AML patient or a B cell comprising a sample from an AML patient with a CD43 peptide or compound of the invention, producing Binding antibodies or B cells against AML, and then obtaining one or more AML-specific domains of AML-specific antibodies or from AML-specific B cells from said AML patient, and providing said one or more structures domain in T cells. Some embodiments provide a method for generating modified T cells comprising immunizing a non-human animal with an inventive CD43 peptide or compound or nucleic acid molecule or functional equivalent or vector, thereby eliciting immunity against AML react, and then obtain one or more AML-specific domains of AML-specific antibodies or AML-specific B cells from the non-human animal, or obtain one or more AML-specific domains encoded as the one or more AML-specific domains or more nucleic acid sequences, optionally after determining whether the AML-specific antibody or AML-specific B cell competes with antibody AT14-013 for binding to CD43, and providing the one or more domains, or the One or more nucleic acid sequences in T cells. Also provided herein is the use of the inventive CD43 peptides or compounds for immunotherapy, and the use of the inventive CD43 peptides for immunotherapy in the context of MHC complexes, and nucleic acid molecules encoding the inventive CD43 peptides or the same. Use of functional equivalents for immunotherapy. Some embodiments provide for the use of a vector comprising an inventive nucleic acid molecule or functional equivalent for immunotherapy. Further provide the use of the inventive CD43 peptide, or the use of a compound comprising the inventive CD43 peptide, or the encoding Use of a nucleic acid molecule or functional equivalent of the inventive CD43 peptide, or use of a vector comprising the inventive nucleic acid molecule or functional equivalent, for the preparation of a drug for myeloproliferative or lymphocyte proliferative diseases, more Preferably AML.

Also provided is an immune composition comprising the inventive CD43 peptide, and/or comprising a compound comprising the inventive CD43 peptide, and/or comprising a nucleic acid molecule encoding the inventive CD43 peptide or a functional equivalent thereof, and/or Or include a vector comprising an inventive nucleic acid molecule or functional equivalent. The immunological composition preferably further comprises biocompatible additives, eg, carriers, diluents, excipients or fillers. Some embodiments provide a vaccine comprising the inventive CD43 peptide, optionally in the context of the MHC complex. Some embodiments provide a vaccine comprising a compound comprising the CD43 peptide of the invention, and a vaccine comprising a nucleic acid molecule encoding the CD43 peptide of the invention or a functional equivalent thereof, and a vaccine comprising a nucleic acid comprising the invention Carriers of molecular or functional equivalents. Other embodiments provide a composition, including the inventive CD43 peptide, or a composition, including a compound comprising the inventive CD43 peptide, or a composition, including a nucleic acid molecule encoding the inventive CD43 peptide or its function, etc. The effector, or a composition, includes a carrier comprising the nucleic acid molecule or functional equivalent of the invention, wherein the composition is a pharmaceutical composition, and further includes a pharmaceutically acceptable carrier, diluent or excipient.

In some embodiments, an isolated, recombinant or purified antibody, or functional portion or functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43 is used in the treatment of myeloproliferative or lymphoproliferative disorders, preferably AML. Antibody AT14-013 was obtained from AML patients in complete remission as described in WO 2015/093949, showing that AT14--013 is effective against AML. In addition, AT14-013 also targets leukemia stem cells, which are known to be more resistant to therapy and often Causes of disease recurrence after treatment. Antibodies that compete with AT14-013 for binding to CD43 would also be very effective against myeloproliferative diseases such as AML. Thus, administration of such antibodies to AML patients will effectively neutralize and/or kill AML cells. Some embodiments thus provide an isolated, recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43 for use as a medicament. Some embodiments provide the use of an isolated, recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43 for the manufacture of a medicament.

Also provided is the use of an isolated, recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with the antibody AT14-013 for binding to CD43, in a method for at least a portion of the treatment or prevention of bone marrow hematopoiesis Myelodysplastic or myeloproliferative or lymphoproliferative diseases, and isolated, recombinant or purified antibodies, or functional parts or functional equivalents thereof, that compete with the antibody AT14-013 for binding to CD43, for use in the preparation of myelodysplastic or Use of the medicament for lymphoproliferative disorders. The myeloproliferative disease preferably includes AML. Still other embodiments provide a composition comprising an isolated, recombinant or purified antibody, or a functional portion or functional equivalent thereof, that competes with antibody AT14-013 for binding to CD43. The composition is preferably a pharmaceutical composition comprising a pharmaceutically acceptable carrier, diluent or excipient.

As previously described, some embodiments provide an isolated, synthesized or recombinant nucleic acid molecule, or functional equivalent thereof, encoding the CD43 peptide of the invention. Such nucleic acid molecules or functional equivalents are, for example, useful in the production of the CD43 peptides of the invention using nucleic acid expression systems, such as host cells. In some embodiments, AML cells are used as host cells. In some embodiments, THP-1 Cells are used as host cells. In some embodiments, cells selected from the group consisting of Kasumi 3 cells, HL60 cells, KG1a cells, SH2 cells, MonoMac6 cells, Molm13 cells, and CML K562 cells are used as host cells.

The nucleic acid molecules or functional equivalents of the invention are also useful for eliciting an immune response. For example, inventive nucleic acid molecules or functional equivalents are administered to AML patients to induce or enhance AML-specific immune responses (immunotherapy). In some embodiments, inventive nucleic acid molecules or functional equivalents are administered to patients with MDS or CML to delay or prevent progression of MDS or CML to AML (prophylactic or semi-prophylactic use). In some embodiments, inventive nucleic acid molecules or functional equivalents are administered to a non-human animal to elicit an anti-AML immune response, from which AML-specific antibodies and/or AML-specific immune cells can then be collected. Alternatively, the variable domains of nucleic acid sequences encoding AML-specific B cells from the non-human animal are sequenced to obtain one or more nucleic acid sequences of the AML-specific variable domains, followed by introduction of AML-specific variable domains comprising AML-specific variable domains One or more nucleic acid molecules of the domain sequence are used in producer cells for the production of AML-specific antibodies.

In some embodiments, an inventive nucleic acid molecule or functional equivalent is present in a gene delivery vehicle that facilitates introduction of the nucleic acid molecule or functional equivalent into a cell of interest. Accordingly, there is further provided a gene delivery vehicle, preferably a vector, comprising the inventive nucleic acid molecule or functional equivalent. Also provided herein is a host cell comprising the inventive nucleic acid molecule or functional equivalent and/or comprising the inventive gene delivery vehicle or vector.

Although the present application may describe features as part of the same embodiment or as part of separate embodiments, the scope of this application also includes some Embodiments that include all or some of the features described herein.

The invention is further explained in the following examples. These examples do not limit the scope of the invention, but merely serve to illustrate the invention.

Example

Example 1

Materials and Methods

Patient and Healthy Human Materials

Research experimental procedures have been approved by the Medical Ethics Committee of the Academic Medical Center. All participants signed informed consent. Participants including healthy individuals and patients with hematologic malignancies were recruited by our clinic who donated peripheral blood and/or bone marrow.

Production of AML-specific clone AT14-013

As described in Example 2 of WO 2015/093949, transduced naive and memory IgG B cells of AML patient 101 were immortalized by introduction of Bcl6 and Bcl-xL as previously described (Kwakkenbos et al. Human, Nat Med 2010 and Example 1 of WO 2015/093949), seeded at a concentration of 20 or 40 cells per well (hereafter referred to as microculture) and seeded with IL-21 and CD40L Amplification. The supernatants of the expanded B cell microcultures were then screened for antibodies binding to AML cell lines (THP-1, MonoMac6, etc.) and to liver and colon cell lines by FACS using human IgG H+L AF647 ( Life Technologies) or human-IgG-PE (Southern Biotech) as secondary antibodies. A number of in-house produced antibodies were used as negative control antibodies, for example, anti-CD30 (expressed on activated B and T lymphocytes), anti-CD33 (expressed on monocytes, myeloid precursor cells, and myeloid leukemias) superior), D25 (for RSV; as described in WO 2008/147196) and AT10-002 (for influenza virus; as described in WO 2013/081463). Microcultures that bound AML cell lines but not liver and colon cell lines were selected and seeded at a concentration of 1 cell/well and their supernatants were re-tested for specificity of AML cell lines. Colonies whose supernatants specifically bound to AML cell lines and did not bind to liver or colon cell lines, or healthy PBMC and bone marrow were selected for sequencing. Colonies were expanded under normal culture conditions in the presence of FBS IgG low serum (Hyclone), and the antibodies purified from the supernatants of these cultures were as described below for recombinant antibodies. The recombinant antibodies were then tested again for specific binding. One of the AML-specific antibodies obtained was AT14-013. The discovered AT14-013 antibody was additionally tested for binding to freshly isolated bud cells from newly diagnosed AML patients (FAB M0-M5) using human IgG H+L AF647 (Life Technologies) as secondary antibody.

Colonization of AML-specific antibody AT14-013

As described in Example 1 of WO 2015/093949, to generate recombinant antibodies, we isolated total RNA with the RNeasy® Mini Kit (Qiagen), generated cDNA, performed PCR and cloned heavy and light chain variable regions into the pCR2.1 TA cloning vector (Invitrogen). To exclude reverse transcriptase or DNA polymerase-induced mutations, we performed multiple independent colonization experiments. To generate recombinant mAbs, we cloned the heavy and light variable regions of each antibody in frame with human IgG1 and kappa (Kappa) constant regions in a pcDNA3.1 (Invitrogen) based vector, and 293T cells were transiently transfected. We purified recombinant antibodies from culture supernatants to Protein A or G, depending on the Ig subtype of the clone.

CDC and ADCC

To quantify complement-dependent cell death (CDC) of target cells induced by the AML-specific antibody AT14-013, we used a FACS-based leukemia cell lysis assay. THP-1 cells were incubated with 2 μM Calcein AM (Becton Dickinson) for 30 minutes at 37°C. Calcein-labeled THP-1 cells were incubated with antibody and rabbit serum complement at 37°C for 4 hours. FACS calibration beads (Accudrop Fluorescent Beads, BD Biosciences) were added to the cells in a 50/50 ratio, after which a standard amount of beads was taken by FACS. Since equal assay volumes have been determined by calibration beads, the amount of dead cells can be calculated as: 100-((Dapi negative, Calcein AM positive cells in each treatment/Dapi negative, Calcein AM positive cells in the control group) ×100). For antibody-dependent cell-mediated cytotoxicity (ADCC), we produced the readout system with Jurkat cells stably transduced with NFAT(6x)-IL2 (minimal promoter)-GFP and CD16a (FcR-IIIa) guide. In this system, NFAT is activated by the bound antibody-activated CD16a receptor, which induces GFP expression, which is then used as a read-out to quantify effector cell activation. AML cells (target cells) were incubated with antibodies and mixed with Jurkat cells (effector cells) stained with Calcein AM as previously described. The effector:target ratio is 1:1.

AT14-013 Target Identification and Confirmation

Solubilized with irrelevant antibodies (in-house produced RSV antibody D25), Protein-G and Streptavidin beads (Pierce) (0,5% Triton X114 supplemented with protease and phosphatase inhibitors (Roche) (Sigma), 150mM NaCl, 10mM Tris-HCL pH7.4,1,5mM MgCl 2) and precleared (preclear) THP-1 cells, a protein to remove nonspecific binding. Precleared cell lysates were then incubated with either bead-bound AML-specific antibody or with influenza virus-specific antibody AT10-002 as a negative control (3 hours at 4°C). Antibody-incubated beads were washed 5 times in lysis buffer supplemented with 0,5% sodium deoxycholate (Deoxycholate) and 0,1% SDS, and bound proteins were eluted from the beads (0, 1 M Glycine pH 10, 5, 150 mM NaCl, 1% Triton X100, 1 mM EDTA) and then electrophoresed on SDS-PAGE. 85% of IP samples were run on SDS-PAGE gels and stained with Imperial Protein Stain (Pierce) for total protein and specific bands were excised for mass spectrometry analysis. The remaining IP samples were run on SDS-PAGE and transferred to PVDF membranes (Bio-RAD) for immunoblotting analysis. Spots were stained by Ponseau S to reveal total protein and blocked with BSA, followed by incubation with mouse-anti-CD43 (colony MEM-59, Abcam) for western blot analysis.

Epitope Mapping: Competition

THP-1 cells were mixed with AT14-013 and commercially available CD43 antibodies: mouse anti-human CD43 PE (Ebioscience; clone 84-3C1), mouse anti-human CD43 FITC (Invitrogen; clone L10), Mouse anti-human CD43 FITC (Abcam; clone MEM-59), mouse anti-human CD43 unlabeled (Abcam; clone MEM-59), mouse anti-human CD43 unlabeled (Thermo Scientific: clone DF -T1), pre-incubated on ice for 60 minutes. The maximum blocking antibody concentration was 10ug/ml. Afterwards, competing antibodies were added at a final concentration of 1 ug/ml. By this step, the final concentration of blocking antibody was 2ug/ml. Cells were incubated on ice for 30 minutes after which dapi (Sigma) was added to exclude dead cells from the analysis. Samples were analyzed by flow cytometry.

Epitope Mapping: Deglycosylation

THP-1 cells were incubated with neuraminidase (Roche; diluted 1:20 or 1:200) for 60 minutes at 37°C to remove sialic acid from CD43 (de Laurentiis et al., 2011). Cells were then washed, blocked in 60% normal goat serum and incubated with AT14-013 and the commercially available CD43 antibodies DF-T1, 84-3C1, L10 and MEM-59 as described above. In order to be able to compare cells stained with different fluorescent dyes, binding to untreated cells (without neuraminidase) was set to 1. Figure 10 shows the fold increase/decrease in binding for neuraminidase-treated cells.

Epitope Mapping: CD43 Truncating Variants

The CD43 cDNA was obtained from Geneart (Life Technologies) and adapted to contain a 3xFLAG tag in-frame on either the C or N-terminus (C-terminus to a signal peptide, including the first 19 of CD43 amino acid). The cDNA was cloned into pHEF-TIG third generation lentiviral vector containing CD43 cDNA 3' to IRES-GFP; VSV-G lentiviral particles were generated in HEK293T cells. THP1, MOLM and other cells were transduced with these viruses in the presence of retronectin and sorted for GFP to obtain a pure population of cells overexpressing CD43. A truncated CD43 variant was established by PCR-cloning of CD43 C-terminal FLAG-tagged cDNA, followed by a wild-type full-length extracellular sequence containing a signal peptide (AA 1-19) (variant A: S20-P400, Followed by 3xFLAG: DYKDHDGDYKDHDIDYKDDDDK) or a truncated extracellular sequence (variant BJ). B: 31-400; C: 59-40; D: 82-400; E: 112-400; F: 133-400; G: 166-400; H: 184-400; I: 202-400; J: 220-400 (transmembrane domain starts at AA 255). These were transformed by lentiviral transduction and GFP sorting. Allogeneic expression in THP1 cells. Sorted cells were lysed and immunoprecipitated with AT14-013 and controls previously described. Eluted IP samples were run on SDS-PAGE and immunoblotted with anti-FLAG-HRP (Sigma) to reveal binding.

result

AT14-013 specifically binds to AML cells

In this example, we identified the target of the AML-specific antibody AT14-013, which was recently developed by our laboratory (WO 2015/093949 and Figure 1). This antibody was derived from a patient designated patient 101. He was diagnosed with intermediate-risk AML (no cytogenetic or molecular abnormalities; FAB classification AML-M5) at age 49. He received two courses of chemotherapy (cytarabine, idarubicine, amsacrine) and one course of consolidation chemotherapy (busulphan, cyclophosphamide) This was followed by autologous hematopoietic stem cell transplantation (HSCT), as no HLA-matched kinship stem cell donor was available. Fourteen months after the initial diagnosis, his disease recurred. He achieved complete remission after one cycle of high-dose cytarabine, after which he received reduced-intensity allogeneic HSCT from a matched, unrelated donor (RIST-MUD). After 6 weeks he developed acute GvHD of the skin, liver and intestine (stage 1; degree II), which responded well to corticosteroid therapy. Given that this patient has remained disease free for 5 years to date, despite the high risk of his disease, this patient can be considered to have developed an effective graft-versus-AML response, and he was selected for the study as an effective AML-specific antibody response s reason. B cells were isolated from a phlebotomy product obtained from a patient 38 months post-HSCT, which were immortalized by introduction of Bcl6 and Bcl-xL as described above (Kwakkenbos et al., Nat Med 2010), and cultured at a concentration of 20 or 40 cells/well. The supernatants of these microcultures were screened for binding to AML cell lines, and microcultures specific for AML were subclone at a concentration of 1 cell/well. One of the antibodies identified by this process was AT14-013, an IgG1 kappa(kappa), highly somatic hypermutated antibody.

AT14-013 binds specifically to multiple AML cell lines and primary AML cells, covering all AML FAB classifications, as shown in Figure 2. In Figure 3, some representative examples of AT14-013 binding to Kasumi3, SH-2, Molm13 and THP-1 and primary leukemia blast cells isolated from newly diagnosed patients are shown. In addition, AT14-013 binds to other myeloid malignancies such as AML from high-risk myeloid hematopoietic syndrome (MDS/RAEB I/II) or blast cell crisis chronic myeloid leukemia (CML) and the CML cell line K562 (section 4). picture). AT14-013 did show partial binding to granule spheres, but not healthy peripheral blood mononuclear cells (PBMC) of the lymphatic lineage, bone marrow, thymocytes, hematological malignancies, or healthy or malignant cells of the liver and colon. AT14-013 did bind to cultured melanocytes and melanoma cell lines (Figure 5).

AT14-013 induces CDC and ADCC in target cells

AT14-013 induced complement-dependent cytotoxicity and antibody-dependent cytotoxicity in AML cell lines and primary isolated AML budding cells (Figure 6).

AT14-013 targets a unique epitope of CD43

We next identified the target of AT14-013. Immunoprecipitation (IP) of THP-1 cell lysates incubated with biotin-labeled sortase-tagged AT14-013 yielded a band of ~140 kDa. The band is specific as it is not seen in THP1 cell lysates AT10-002 IP or Jurkat cell lysate IP (Figure 7). Mass spectrometry analysis of immunoprecipitated bands revealed CD43 as the target protein. Three expected intracellular peptides were identified, resulting in 7% protein coverage, no extracellular peptides were identified due to these being highly glycosylated. AT14-013 binding to CD43 was confirmed by Western blot analysis. Briefly, THP-1 and Molm13 cell lysates were immunoprecipitated with AT14-013 or the influenza virus specific antibody AT10-002. Western blot analysis of mouse-anti-CD43 (clone Mem59) confirmed CD43 as the binding target of AT14-013 (Figure 8).

CD43 is widely expressed on healthy and malignant cells. CD43-specific antibodies have been produced and are commercially available, eg, DF-T1, 84-3C1, L10, and MEM-59. With these antibodies, we confirmed CD43 expression by THP-1 cells (Fig. 9a). AT14-013 did not bind to non-myeloid cells, and the observation of the different binding profiles (Figure 9b) of AT14-013 to various cells and cell lines compared to other CD43 antibodies showed that compared to other CD43 antibodies, AT14-013 recognizes different CD43 epitopes. Indeed, when we performed competition experiments with THP-1 cells incubated with commercially available CD43 antibodies and AT14-013, we found that these CD43 antibodies competed with each other for binding to THP-1, but not with AT14-013 (Figs. 9c and 9d). Notably, CD43 clones L10 and 84-3C1 have been described to compete with each other (L. Borche et al., 2005); this was also confirmed in our experiments.

The CD43 protein is a highly glycosylated protein (de Laurentiis et al., 2011). The CD43 antibodies Mem59, DF-T1, and 84-3C1 (but not L10) bind to sialylated epitopes, since pretreatment of target cells with neuraminidase removes α-N-acetylneuraminic acid (sialic acid), these antibodies are lost Binding to CD43 (US2010/0234562A1). In Figure 10, we demonstrate that AT14-013 binding to THP-1 cells is also lost after pre-incubation of THP-1 cells with neuraminidase, showing that AT14-013 specifically binds to the epitope of CD43 sialylation base.

To more specifically identify the binding epitope of AT14-013, we generated 10 Flag-tagged extracellular truncated variants of CD43 expressed in HEK and THP1 cells. Western blot analysis of cell lysates from these cells incubated with Mem59 or DF-T1 confirmed that these antibodies bind to similar epitopes located between amino acids 59-82 (panels 11a and 11b). We tested AT14-013 binding by immunoprecipitation of THP1 cells transduced with these truncated variants. AT14-013 reacted strongly with variants A-F, to a lesser extent with variant G, and did not react with variants H-J, as shown by anti-Flag immunoblotting analysis of IP (panels 12a and 12b). In Figure 12c, we confirmed the IP of AT14-013 with an anti-C-terminal CD43 antibody. Endogenous CD43 was present in all samples, whereas truncated CD43 was present only up to variant G. Therefore, we concluded that the epitope of AT14-013 is between amino acids 133 and 184.

Example 2: Binding to AML bud cells

Materials and Methods

Antibody AT14-013 was tested for binding to different cells using the method described in Example 1 under the heading "Production of AML-specific clone AT14-013". Patient samples were stained with anti-human CD45 (BD) prior to analysis. AML cells were defined as CD45dim. Healthy PBMCs were stained with anti-human CD3 (biolegend). Polymorphonuclear cells derived from tonsils were isolated by ficol density gradient.

result

AT14-013 specifically binds to a variety of AML cell lines and primary AML cells, covering all AML FAB classifications, as shown in Example 1 and Figure 4. In addition, we tested the antibody on a wider range of AML budding cells. It was shown to bind to all AML budding cells tested so far, and was generally better than commercial anti-CD43 antibodies. Interestingly, the sialic acid independent L10 antibody bound the least in almost all samples. In addition, antibodies were tested on healthy CD43-expressing T cells and cells derived from tonsils. Here, only commercial antibodies were shown to be stained. The results are summarized in Figure 14.

Example 3: ADCC and CDC

In addition to Example 1 and Figure 6, another ADCC and CDC experiment was further carried out.

Materials and Methods

To quantify antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cell death (CDC) of target cells induced by the AML-specific antibody AT14-013, we used a FACS-based leukemia cell lysis assay. SH2 cells were incubated with 10 nM Calcein AM (Becton Dickinson) for 30 minutes at 37°C. Calcein-labeled cells were incubated with antibody and healthy peripheral blood mononuclear cells (PBMC; effector:target 50:1) for 4 hours or with rabbit serum complement for 1 hour at 37°C. FACS calibration beads (Accudrop Fluorescent Beads, BD Biosciences) were added to the cells in a 50/50 ratio, after which a standard amount of beads was taken by FACS. Since equal assay volumes have been determined by calibration beads, the amount of dead cells can be calculated as: 100-((Dapi negative, Calcein AM positive cells in each treatment cells/Dapi negative, Calcein AM positive cells in the control group) × 100).

result

AT14-013 induces CDC and ADCC in target cells

AT14-013 induced antibody-dependent cytotoxicity (Fig. 15A) and complement-dependent cytotoxicity (Fig. 15B) in AML cell lines and primary isolated AML budding cells.

Example 4: Epitope Mapping: CD43 Truncating Variants

In addition to Example 1 and Figure 12, the binding epitope of AT14-013 was further investigated.

Materials and Methods

The same method as in Example 1 was used. The CD43 cDNA was obtained from Geneart (Life Technologies) and adapted to contain the 3xFLAG tag in-frame on either the C or N-terminus (C-terminus to the signal peptide, including the first 19 of CD43 amino acid). The cDNA was cloned into pHEF-TIG third generation lentiviral vector containing CD43 cDNA 3' to IRES-GFP; VSV-G lentiviral particles were generated in HEK293T cells. THP1, MOLM and other cells were transduced with these viruses in the presence of retronectin and sorted for GFP to obtain a pure population of CD43 transduced cells. A truncated CD43 variant was established by PCR-cloning of the C-terminal FLAG-tagged cDNA of CD43 containing the signal peptide (AA 1-19) followed by the wild-type full-length extracellular sequence (variant A: S20-P400 , followed by 3xFLAG: DYKDHDGDYKDHDIDYKDDDDK) or a truncated extracellular sequence (variant BJ). B: 31-400; C: 59-400; D: 82-400; E: 112-400; F: 133-400; F2: 148-400; G: 166-400; H: 184-400; I: 202-400; J: 220-400 (transmembrane domain starts at AA 255). These variants were expressed in THP1 cells by lentiviral transduction and GFP sorting. Sorted cells were lysed and immunoprecipitated with AT14-013 and controls previously described. Eluted IP samples were run on SDS-PAGE and immunoblotted with anti-FLAG-HRP (Sigma) to reveal binding.

result

AT14-013 targets a unique epitope of CD43

To more specifically identify the binding epitope of AT14-013, we generated 11 Flag-tagged extracellular truncated variants of CD43 expressed in HEK and THP1 cells. We tested AT14-013 binding by immunoprecipitation of THP1 cells transduced with these truncated variants. AT14-013 reacted strongly with variant AF, to a lesser extent with variant F2, to a lesser extent with variant G, and did not react with variant HJ, as shown by anti-Flag immunoblotting analysis of IP (Figs. 16a and 16b). In panel 16b, we confirmed the IP of AT14-013 with anti-C-terminal CD43 antibody. Endogenous CD43 was present in all samples, whereas truncated CD43 was present only up to variant F2. Therefore, we concluded that the epitope of AT14-013 includes one or more amino acid residues, which are present between amino acids 133 and 165. Given that AT14-013 reacts to a lesser extent with variant F2 (starting at amino acid position 148, as shown in Figure 13), we also conclude that the epitope of AT14-013 includes one or more amino acid residues base, which exists between amino acids 133 and 147.

Example 5: AT14-013 inhibits AML in vivo ( in vivo )

Currently known experimental procedures are described, for example, in Miller et al., Blood (2013), Vol. 121, No. 5, e1-e4.

To assess the efficacy of AT14-013 against AML in vivo , humanized mice reconstituted with human hematopoietic cells and xenografted with SH2 cells were treated. By injecting 50 000 CD34 ⁺ CD38 ^- hematopoietic stem cells in newborn not deadly radiation (sublethaly irradiated newborn) (1-5 days old) liver, humanized 6 female ^{NOD.Cg-Prkdc scid} I12rg tm1Wj1 ^{/ SzJ} (NSG, The Jackson Laboratory). At 8 weeks, mice were bled to assess engraftment of human hematopoietic cells in their blood. In this experiment, only mice with greater than 20% human chimerism in peripheral blood were used. 6 mice 5 meet this criteria, and inoculated with 10x10 ⁶ Expression of the luciferase enzyme GFP d0 SH-2 cells intravenously. On d14, mice were IP injected with luciferin (150 mg/kg) and tumor engraftment was detected by in vivo bioluminescence. According to this measurement, mice were randomly divided into 2 groups and then vaccinated by iv with AT14-013 or antibody AT10-002 (against influenza virus, as described in WO 2013/081463, as control group) (375 μg) Dosing twice a week. Bioluminescence was measured weekly as previously described. On d39, mice were sacrificed by cervical dislocation under deep anesthesia, and the organs were exposed and bioluminescence was quantified. Single cell suspensions of liver and bone marrow were obtained and the presence of SH-2 GFP+ cells was quantified by FACS. Treatment of mice engrafted with SH-2 AML cells resulted in 90.3% inhibition of tumor growth as measured by whole body measurements at the time of sacrifice (p<0.001, repeat ANOVA, Figure 17A). The number of AML cells, as measured by photons per minute (cpm), showed a substantial reduction in all organs measured (p=0.0011, repeated two-factor ANOVA, Figure 17B). This observation was confirmed by FACS assessment of tumor cell numbers in bone marrow and liver (p=0.0017, two-factor ANOVA, Figure 17C).

Therefore, the anti-species specific for the CD43 peptide of the present invention It is additionally suitable for use in the in vivo treatment or prevention of myeloproliferative or lymphoproliferative disorders such as AML.

Example 6

Materials and Methods

Fetal liver, bone marrow and thymus tissue were obtained between the 16th and 21st week of pregnancy from the His Mouse Facility at AMC (under Dutch law: Wet Foetaal Weefsel). A CD34-enriched monocyte suspension was obtained by whole-organ Stomacher cell preparation followed by density gradient centrifugation and magnetic bead separation. A CD34-enriched monocyte suspension of fetal bone marrow was prepared by density gradient centrifugation and magnetic bead separation.

Binding of antibody AT14-013 to cells from fetal liver, fetal thymus and fetal bone marrow was measured by flow cytometry using commercially available CD34 (BD, cat. 343516) and CD38 (BD, cat. 303522) antibodies to distinguish different subsets of these samples.

result

AT14-013 specifically binds to the oncofetal epitope of CD43

As previously described, AT14-013 is a CD43-specific antibody that recognizes a unique, onco-sialylated tumor antigen expressed primarily by AML and MDS bud cells. Tumor antigens are either abnormal proteins with tumor-specific expression or abnormally expressed normal cells, such as carcinoembryonic antigens, which are normally antigens expressed only by fetal tissue during ontogeny.

Neoplastic transformation of cells is often associated with the expression of oncofetal antigens. We found that CD34+ CD38- bone marrow stem cells obtained from fetal liver and fetal bone marrow expressed the AT14-013 epitope of CD43, However, CD34+ CD38+ precursor cells or CD34- CD38- mature cells obtained from fetal liver and fetal bone marrow did not express (Fig. 18). These results show that AT14-013 is capable of binding the cancer-sialylated epitope of CD43, which is widely expressed by AML and MDS in adults.

Example 7

Donor #101 (same as the donor who obtained AT14-013-producing B cells) AML budding cells with AT14-013 and with antibodies specific for CD34 and CD38 (same procedure as in Example 6), and with Antibodies against CD45 (BD, cat 348815) were stained together to identify the overall blast cell population (CD45 dim) and healthy cells in the bone marrow and analyzed by flow cytometry (Figure 19). This shows that AT14-013 binds to the patient's leukemia bud cells, which are found in, including leukemia stem cells, defined as CD34+CD38- bud cells.

It was thus concluded that the antibody AT14-013 reacts with autologous leukemia stem cells, making AT14-013 particularly suitable for use in the treatment or prevention of myeloproliferative or lymphoproliferative disorders, since it also targets leukemia stem cells, which are known to be relatively Treatment-resistant and often the cause of disease recurrence after treatment.

Here, followed by another antibody, which is specific for the CD43 peptide of the invention, for example, an antibody that competes with the antibody AT14-013 for binding to CD43, is also particularly suitable for the treatment or prevention of myeloproliferative or lymphoproliferative diseases .

references

Bennett, J.M. et al., 1976. Proposals for the classification of the acute leukaemias. French-American-British (FAB) co-operative group. British Journal of Haematology, 33(4), pp.451-458.

Borche, L. et al., 2005. CD43 monoclonal antibodies recognize the large sialoglycoprotein of human leukocytes. European Journal of Immunology, 17(10), pp 1523-1526

European Patent Application No. 1974017

Hanly et al., Review of polyclonal antibody production procedures in mammals and poultry. ILAR Journal (1995); Vol.37, Number 3: 93-118

International Patent Application No. WO 2015/093949

International Patent Application No. WO 2006/121240

International Patent Application No. WO 2007/146172

Kim et al., Characterization of two novel mAbs recognizing different epitopess on CD43. Immune Network (2014). Vol. 14, No. 3: 164-170

Kwakkenbos MJ et al., Generation of stable monoclonal antibody-producing B cell receptor-positive human memory B cells by genetic programming. Nat Med. 2010. 16(1):123-8..

de Laurentiis, A. et al., 2011. Mass Spectrometry-Based Identification Of The Tumor Antigen UN1 as the Transmembrane CD43 Sialoglycoprotein. Molecular & Cellular Proteomics, 10(5), pp.M111.007898-M111.007898

Malcovati, L. et al., 2013. Diagnosis and treatment of primary myelodysplastic syndromes in adults: recommendations from the European LeukemiaNet. Blood, 122(17), pp.2943-2964

Miller et al. , Blood (2013), Vol.121, No.5, e1-e4

Schmid K, Hediger MA, Brossmer R, et al., Amino acid sequence of human plasma galactoglycoprotein: identity with the extracellular region of CD43 (sialophorin). Proc. Natl. Acad. Sci. USA 1992;89(2):663- 667

Shelley et al.,Molecular characterization of sialophorin (CD43), the lymphocyte surface sialoglycoprotein defective in Wiskott-Aldrich syndrome. Proc. Natl. Acad. Sci. USA 1989; Vol. 86: 2819-2823

Swerdlow S.H. WHO classification of Tumours of Haematopoietic and Lymphoid Tissues. International Agency for Research on Cancer, 2008. ISBN: 978-92-832-2431-0

Tuccillo et al., Cancer-associated CD43 glycoforms as target of immunotherapy. Mol. Cancer ther. (2014a) 13(3): 752-762

Tuccillo et al., Aberrant glycosylation as biomarker for cancer: focus on CD43. BioMed research International (2014b) Article ID 742831, 13 pages. http://dx.doi.org/10.1155/2014/742831

US Patent Application No. 9,005,974

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<213> Artificial sequences

<220>

<223> Synthetic Constructs

<400> 25

<210> 26

<211> 51

<212> DNA

<213> Artificial sequences

<220>

<223> Light chain CDR1

<220>

<221> CDS

<222> (1)..(51)

<400> 26

<210> 27

<211> 17

<212> PRT

<213> Artificial sequences

<220>

<223> Synthetic Constructs

<400> 27

<210> 28

<211> 45

<212> DNA

<213> Artificial sequences

<220>

<223> Light chain Fw2

<220>

<221> CDS

<222> (1)..(45)

<400> 28

<210> 29

<211> 15

<212> PRT

<213> Artificial sequences

<220>

<223> Synthetic Constructs

<400> 29

<210> 30

<211> 21

<212> DNA

<213> Artificial sequences

<220>

<223> Light chain CDR2

<220>

<221> CDS

<222> (1)..(21)

<400> 30

<210> 31

<211> 7

<212> PRT

<213> Artificial sequences

<220>

<223> Synthetic Constructs

<400> 31

<210> 32

<211> 96

<212> DNA

<213> Artificial sequences

<220>

<223> Light chain Fw3

<220>

<221> CDS

<222> (1)..(96)

<400> 32

<210> 33

<211> 32

<212> PRT

<213> Artificial sequences

<220>

<223> Synthetic Constructs

<400> 33

<210> 34

<211> 27

<212> DNA

<213> Artificial sequences

<220>

<223> Light chain CDR3

<220>

<221> CDS

<222> (1)..(27)

<400> 34

<210> 35

<211> 9

<212> PRT

<213> Artificial sequences

<220>

<223> Synthetic Constructs

<400> 35

<210> 36

<211> 30

<212> DNA

<213> Artificial sequences

<220>

<223> Light chain Fw4

<220>

<221> CDS

<222> (1)..(30)

<400> 36

<210> 37

<211> 10

<212> PRT

<213> Artificial sequences

<220>

<223> Synthetic Constructs

<400> 37

Claims (14)

An isolated, recombinant or purified CD43 peptide having a length of up to 100 amino acid residues, wherein the peptide comprises an amino acid sequence GTITTNSPETSSRTSGAPVTTAASSLETSRGTS, and wherein the peptide has an AML-specific glycosylation form or MDS-specific glycosylated forms. A compound comprising the CD43 peptide as described in item 1 of the patent application scope. An isolated, synthesized or recombinant nucleic acid molecule encoding the CD43 peptide described in item 1 of the scope of the application. A vector, comprising the nucleic acid molecule as described in item 3 of the patent application scope. A CD43 peptide as described in item 1 of the patented scope, a compound as described in item 2 of the patented scope, nucleic acid molecule as described in item 3 of the patented scope, or as described in item 4 of the patented scope of application The use of the vector is used for preparing a drug for inducing an immune cell and/or an antibody against the CD43 peptide. A CD43 peptide as described in item 1 of the patented scope, a compound as described in item 2 of the patented scope, nucleic acid molecule as described in item 3 of the patented scope, or as described in item 4 of the patented scope of application The use of the carrier is for in vitro binding, detecting and/or obtaining an immune cell and/or an antibody that can specifically bind to myeloproliferative cells or lymphoplastic cells. The use according to claim 6, wherein the myeloproliferative cells are acute myeloid leukemia (AML) cells, myelodysplastic syndrome (MDS) cells, or chronic myeloid leukemia (CML) cells. An immune composition, comprising CD43 as described in item 1 of the patent application scope Peptides, compounds as described in item 2 of the claimed scope, nucleic acid molecules as described in item 3 of the claimed scope, or carriers as described in claim 4 of the claimed scope. The immune composition according to item 8 of the claimed scope, wherein the composition is a pharmaceutical composition, which also includes a pharmaceutically acceptable carrier, diluent or excipient. A diagnostic kit, comprising the CD43 peptide as described in item 1 of the patented scope, or the compound as described in item 2 of the patented scope of application, and a tool for detecting antibodies or immune cells. A CD43 peptide as described in item 1 of the patented scope, a compound as described in item 2 of the patented scope, nucleic acid molecule as described in item 3 of the patented scope, or as described in item 4 of the patented scope of application The use of the carrier is for the preparation of a prophylactic agent for MDS, AML or CML patients. A CD43 peptide as described in item 1 of the patented scope, a compound as described in item 2 of the patented scope, nucleic acid molecule as described in item 3 of the patented scope, or as described in item 4 of the patented scope of application The use of the carrier is for preparing medicines for treating myeloproliferative cell or lymphocyte proliferative cell diseases. A use of the CD43 peptide according to the first item of the patent application, or the compound according to the second item of the patent application, for preparing a diagnostic reagent for diagnosing MDS, AML or CML. A host cell, comprising the nucleic acid molecule as described in item 3 of the patented scope, or the vector as described in item 4 of the patented scope of application.

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